Composition of tumor-associated peptides and related anti-cancer vaccine for the treatment of gastric cancer and other cancers

ABSTRACT

The present invention relates to immunotherapeutic peptides and their use in immunotherapy, in particular the immunotherapy of cancer. The present invention discloses tumor-associated T-helper cell peptide epitopes, alone or in combination with other tumor-associated peptides that serve as active pharmaceutical ingredients of vaccine compositions which stimulate anti-tumor immune responses. In particular, the composition of the peptides of the present invention can be used in vaccine compositions for eliciting anti-tumor immune responses against gastric cancers (GC).

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application61/315,715, filed Mar. 19, 2010, and UK patent application GB1004575.5,filed on Mar. 19, 2010, the entire contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to immunotherapeutic peptides and theiruse in immunotherapy, in particular the immunotherapy of cancer. Thepresent invention discloses tumor-associated T-helper cell peptideepitopes, alone or in combination with other tumor-associated peptidesthat serve as active pharmaceutical ingredients of vaccine compositionswhich stimulate anti-tumor immune responses. In particular, thecomposition of the peptides of the present invention can be used invaccine compositions for eliciting anti-tumor immune responses againstgastric and other cancers (GC).

For the purposes of the present invention, all references as citedherein are incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

Gastric cancer is a disease in which malignant cells form in the liningof the stomach. Stomach or gastric cancer can develop in any part of thestomach and may spread throughout the stomach and to other organs;particularly the esophagus, lungs and the liver. Stomach cancer is thefourth most common cancer worldwide with 930,000 cases diagnosed in2002. It is a disease with a high death rate (˜800,000 per year) makingit the second most common cause of cancer death worldwide after lungcancer. It is more common in men and occurs more often in Asiancountries and in developing countries. (Informations can be obtainedfrom the WHO.)

It represents roughly 2% (25,500 cases) of all new cancer cases yearlyin the United States, but it is more common in other countries. It isthe leading cancer type in Korea, with 20.8% of malignant neoplasms. InJapan gastric cancer remains the most common cancer for men. Each yearin the United States, about 13,000 men and 8,000 women are diagnosedwith stomach cancer. Most are over 70 years old.

Stomach cancer is the fourth most common cancer worldwide, after cancersof the lung, breast, and colon and rectum. Furthermore, stomach cancerremains the second most common cause of death from cancer. The AmericanCancer Society estimates that in 2007 there were an estimated onemillion new cases, nearly 70% of them in developing countries, and about800,000 deaths (see the publications of the American Cancer Society).

Tremendous geographic variation exists in the incidence of this diseasearound the world. Rates of the disease are highest in Asia and parts ofSouth America and lowest in North America. The highest death rates arerecorded in Chile, Japan, South America, and the former Soviet Union.

Gastric cancer is often diagnosed at an advanced stage, becausescreening is not performed in most of the world, except in Japan (and ina limited fashion in Korea) where early detection is often done. Thus,it continues to pose a major challenge for healthcare professionals.Risk factors for gastric cancer are Helicobacter pylori (H. pylori)infection, smoking, high salt intake, and other dietary factors. A fewgastric cancers (1% to 3%) are associated with inherited gastric cancerpredisposition syndromes. E-cadherin mutations occur in approximately25% of families with an autosomal dominant predisposition to diffusetype gastric cancers. This subset of gastric cancer has been termedhereditary diffuse gastric cancer.12 It may be useful to provide geneticcounseling and to consider prophylactic gastrectomy in young,asymptomatic carriers of germ-line truncating

The wall of the stomach is made up of 3 layers of tissue: the mucosal(innermost) layer, the muscularis (middle) layer, and the serosal(outermost) layer. Gastric cancer begins in the cells lining the mucosallayer and spreads through the outer layers as it grows. Four types ofstandard treatment are used. Treatment for gastric cancer may involveSurgery, Chemotherapy, Radiation therapy or Chemoradiation. Surgery isthe primary treatment for gastric cancer. The goal of surgery is toaccomplish a complete resection with negative margins (R0 resection).However, approximately 50% of patients with locoregional gastric cancercannot undergo an R0 resection. R1 indicates microscopic residual cancer(positive margins); and R2 indicates gross (macroscopic) residual cancerbut not distant disease. Patient outcome depends on the initial stage ofthe cancer at diagnosis. (NCCN Clinical Practice Guidelines inOncology™)

The 5-year survival rate for curative surgical resection ranges from30-50% for patients with stage 11 disease and from 10-25% for patientswith stage III disease. These patients have a high likelihood of localand systemic relapse. Metastasis occurs in 80-90% of individuals withstomach cancer, with a six month survival rate of 65% in those diagnosedin early stages and less than 15% of those diagnosed in late stages.

There thus remains a need for new efficacious and safe treatment optionfor gastric cancer, prostate carcinoma, oral cavity carcinomas, oralsquamous carcinoma (OSCC), acute myeloid leukemia (AML) (Qian et al.,2009), H. pylori-induced MALT lymphoma (Banerjee et al., 2000), coloncarcinoma/colorectal cancer, glioblastoma, non-small-cell lung cancer(NSCLC), cervical carcinoma, human breast cancer, prostate cancer, coloncancer, pancreatic cancers, pancreatic ductal adenocarcinoma, ovariancancer, hepatocellular carcinoma, liver cancer, brain tumors ofdifferent phenotypes, leukemias such as acute lymphoblastic leukemia(ALL), lung cancer, Ewing's sarcoma, endometrial cancer, head and necksquamous cell carcinoma, epithelial cancer of the larynx, oesophagealcarcinoma, oral carcinoma, carcinoma of the urinary bladder, ovariancarcinomas, renal cell carcinoma, atypical meningioma, papillary thyroidcarcinoma, brain tumors, salivary duct carcinoma, cervical cancer,extranodal T/NK-cell lymphomas, Non-Hodgkins Lymphoma and malignantsolid tumors of the lung and breast and other tumors. There also remainsa need for treatment options that enhance the well-being of the patientswithout using chemotherapeutic agents or other agents which may lead tosevere side effects.

Colorectal Carcinoma

According to the American Cancer Society, colorectal cancer (CRC) is thethird most common cancer in the US, afflicting more than 175,000 newpatients each year. In the US, Japan, France, Germany, Italy Spain andthe UK, it affects more than 480,000 patients. It is one of the mostcommon causes of cancer mortality in developed countries. Researchsuggests that the onset of colorectal cancer is the result ofinteractions between inherited and environmental factors. In most casesadenomatous polyps appear to be precursors to colorectal tumors; howeverthe transition may take many years. The primary risk factor forcolorectal cancer is age, with 90% of cases diagnosed over the age of 50years. Other risk factors for colorectal cancer according to theAmerican Cancer Society include alcohol consumption, a diet high in fatand/or red meat and an inadequate intake of fruits and vegetables.Incidence continues to rise, especially in areas such as Japan, wherethe adoption of westernized diets with excess fat and meat intake and adecrease in fiber intake may be to blame. However, incidence rates arerising not as fast as previously which may be due to increasingscreening and polyp removal, thus preventing progression of polyps tocancer.

As in most solid tumors, first line treatment is surgery, however, itsbenefits remain confined to early-stage patients, yet a significantproportion of patients is diagnosed in advanced stages of the disease.For advanced colorectal cancer chemotherapy regimens based onfluorouracil-based regimens are standard of care. The majority of theseregimens are the so-called FOLFOX (infusional 5-FU/leucovorin plusoxaliplatin) and FOLFIRI (irinotecan, leucovorin, bolus andcontinuous-infusion 5-FU) protocols.

The introduction of third-generation cytotoxics such as irinotecan andoxaliplatin has raised the hope of significantly improving efficacy, butprognosis is still relatively poor, and the survival rate generallyremains at approximately 20 months in metastatic disease and, as aresult, the unmet needs in the disease remain high.

Recently, a novel generation of drugs, molecular-targeted agents, suchas AVASTIN® (bevacizumab) and ERBITUX® (cetuximab), became available,and about 40 compounds are in late-stage clinical development fordifferent stages of colorectal cancer. Combinations of several of thesecompounds increase the number of potential treatment options to beexpected for the future. The vast majority of substances is in phase 2,with EGFR addressed by these compounds more often than by any other drugin development for colorectal cancer, which is due to the fact that in^(˜)80% of patients with colorectal cancer EGFR expression isupregulated.

Clinical trials with stage II patients combining chemotherapy with therecently approved monoclonal antibodies (mAbs) (cetuximab+irinotecan orFOLFOX4; bevacizumab as a single-agent or together with FOLFOX4) arecurrently conducted. Three to four year observation periods are expectedfor statistically significant results from these trials.

Monoclonal antibodies (mAbs) presently used in oncology in general havean excellent chance of not interfering with active immunotherapy. Infact, there is preclinical evidence suggesting that depletion of VEGF(by bevacizumab) contributes positively to DC-mediated activation ofT-cells.

Prostate Carcinoma and Other Exemplary Tumors

With an estimated 27,050 deaths in 2007, prostate cancer is a leadingcause of cancer death in men. Although death rates have been decliningamong white and African American men since the early 1990s, rates inAfrican American men remain more than twice as high as those in whitemen. Prostate cancer is the most frequently diagnosed cancer in men. Forreasons that remain unclear, incidence rates are significantly higher inAfrican American men than in white men. Incidence rates of prostatecancer have changed substantially over the last 20 years: rapidlyincreasing from 1988-1992, declining sharply from 1992-1995, andincreasing modestly since 1995. These trends in large part reflectincreased prostate cancer screening with the prostate-specific antigen(PSA) blood test. Moderate incidence increases in the last decade aremost likely attributable to widespread PSA screening among men youngerthan 65. Prostate cancer incidence rates have leveled off in men aged 65years and older. Rates peaked in white men in 1992 (237.6 per 100,000men) and in African American men in 1993 (342.8 per 100,000 men).

Treatment for prostate cancer may involve watchful waiting, surgery,radiation therapy, High Intensity Focused Ultrasound (HIFU),chemotherapy, cryosurgery, hormonal therapy, or some combination of theabove. The best option depends on the stage of the disease, the Gleasonscore, and the PSA level. Other important factors include the man's age,his general health, and his feelings about potential treatments andtheir possible side effects. Because all treatments can have significantside effects, such as erectile dysfunction and urinary incontinence,treatment discussions often focus on balancing the goals of therapy withthe risks of lifestyle alterations.

If the cancer has spread beyond the prostate, treatment optionssignificantly change, so most doctors who treat prostate cancer use avariety of tomograms to predict the probability of spread. Treatment bywatchful waiting, HIFU, radiation therapy, cryosurgery, and surgery aregenerally offered to men whose cancer remains within the prostate.Hormonal therapy and chemotherapy are often reserved for disease whichhas spread beyond the prostate. However, there are exceptions: radiationtherapy may be used for some advanced tumors, and hormonal therapy isused for some early stage tumors. Cryotherapy, hormonal therapy, andchemotherapy may also be offered if initial treatment fails and thecancer progresses.

In a significant number of patients with prostate carcinoma who undergoradical prostatectomy because of clinically suspected organ-limitedgrowth, a definitive histological workup of the surgical preparationshows a locally extensive tumor extending beyond the borders of theorgan. These patients have a high risk for early local recurrence,usually detectable as an increasing PSA level in terms of a biochemicalrelapse. Therapeutic options in this situation include externalradiotherapy and hormone ablation; however, the value of thesetherapeutic approaches, especially with respect to prolonging thepatient's long-term survival, must not be regarded as proven. Inaddition, possible treatment-associated complications such as thedevelopment of urethral strictures (radiotherapy), loss of libido andimpotence, the risk of a reduction in skeletal calcium salts in terms ofosteoporosis, and a markedly increased risk of pathologic bone fractures(hormone ablation) must be considered.

More than 90% of all prostate cancers are discovered in the local andregional stages; the 5-year relative survival rate for patients whosetumors are diagnosed at these stages approaches 100%. Over the past 25years, the 5-year survival rate for all stages combined has increasedfrom 69% to nearly 90%. According to the most recent data, relative10-year survival is 93% and 15-year survival is 77%. The dramaticimprovements in survival, particularly at 5 years, are partlyattributable to earlier diagnosis and improvements in treatment.Nevertheless, the survival rate drops significantly after the spreadingto other tissues and organs.

Lung Cancer

An estimated 210,000 new cases are expected in 2007 in the USA,accounting for about 15% of cancer diagnoses. The incidence rate isdeclining significantly in men, from a high of 102 cases per 100,000 in1984 to 78.5 in 2003. In women, the rate is approaching a plateau aftera long period of increase. Lung cancer is classified clinically as smallcell (13%) or non-small cell (87%) for the purposes of treatment.

Lung cancer accounts for the most cancer-related deaths in both men andwomen. An estimated 160,390 deaths, accounting for about 29% of allcancer deaths, are expected to occur in 2007. Since 1987, more womenhave died each year from lung cancer than from breast cancer. Deathrates have continued to decline significantly in men from 1991-2003 byabout 1.9% per year. Female lung cancer death rates are approaching aplateau after continuously increasing for several decades. These trendsin lung cancer mortality reflect the decrease in smoking rates over thepast 30 years.

Treatment options are determined by the type (small cell or non-smallcell) and stage of cancer and include surgery, radiation therapy,chemotherapy, and targeted biological therapies such as bevacizumab(Avastin®) and erlotinib (Tarceva®). For localized cancers, surgery isusually the treatment of choice. Recent studies indicate that survivalwith early-stage, non-small cell lung cancer is improved by chemotherapyfollowing surgery. Because the disease has usually spread by the time itis discovered, radiation therapy and chemotherapy are often used,sometimes in combination with surgery. Chemotherapy alone or combinedwith radiation is the usual treatment of choice for small cell lungcancer; on this regimen, a large percentage of patients experienceremission, which is long lasting in some cases.

The 1-year relative survival for lung cancer has slightly increased from37% in 1975-1979 to 42% in 2002, largely due to improvements in surgicaltechniques and combined therapies. However, the 5-year survival rate forall stages combined is only 16%. The survival rate is 49% for casesdetected when the disease is still localized; however, only 16% of lungcancers are diagnosed at this early stage.

TABLE 1 Estimated new cancer cases and deaths by sex for the US in 2007(American Cancer Society. Cancer Facts & Figures 2007. Atlanta: AmericanCancer Society; 2007.) Estimated New Cases Both Estimated Deaths SitesSexes Male Female Both Sexes Male Female Glioma and 20,500 11,170 9,33012,740 7,150 5,590 Brain Breast 180,510 2,030 178,480 40,910 450 40,460Prostate 218,890 218,890 27,050 27,050 Esophagus 15,560 12,130 3,43013,940 10,900 3,040 Colon 112,340 55,290 57,050 52,180 26,000 26,180Renal 51,190 31,590 19,600 12,890 8,080 4,810 Pancreas 37,170 18,83018,340 33,370 16,840 16,530 Squamous cell 1,000,000 n.d. n.d. n.d. n.d.n.d. carcinomas; Keratinocytic neoplasms of the skin Leukemia 44,24024,800 19,440 21,790 12,320 9,470 Lung 213,380 114,760 98,620 160,39089,510 70,880 Non-Hodgkins 63,190 34,210 28,990 18,660 9,600 9,060Lymphoma Ovarian 22,430 22,430 15,280 15,280 Melanoma 59,940 33,91026,030 8,110 5,220 2,890

There thus remains a need for new efficacious and safe treatment optionfor glioblastoma, prostate tumor, breast cancer, esophageal cancer,colorectal cancer, clear cell renal cell carcinoma, lung cancer, CNS,ovarian, melanoma, pancreatic cancer, squamous cell carcinoma, leukemiaand medulloblastoma and other tumors which show an overexpression ofsurvivin, enhancing the well-being of the patients without usingchemotherapeutic agents or other agents which may lead to severe sideeffects.

SUMMARY OF THE INVENTION

In the present invention, the inventors isolated and characterizedpeptides binding to HLA class I or II molecules directly from mammaliantumors, i.e. primary samples of mainly gastric cancer patients, but alsofrom primary tissue samples of glioblastoma, colorectal cancers, renalcell carcinoma, lung cancers, pancreatic cancers, malignant melanoma,and cancer of the stomach.

The present invention provides peptides that stem from antigensassociated with tumorigenesis, and have the ability to bind sufficientlyto MHC(HLA) class II molecules for triggering an immune response ofhuman leukocytes, especially lymphocytes, especially T lymphocytes,especially CD4-positive T lymphocytes, especially CD4-positive Tlymphocytes mediating T_(H1)-type immune responses.

The present invention also provides peptides that stem from antigensassociated with tumorigenesis, and have the ability to bind sufficientlyto MHC(HLA) class I molecules for triggering an immune response of humanleukocytes, especially lymphocytes, especially T lymphocytes, especiallyCD8-positive cytotoxic T-lymphocytes as well as combinations of the twothat are particularly useful for vaccination of patients that sufferfrom cancer.

The present invention also provides a pharmaceutical compositioncomprising at least two peptides containing an amino acid sequenceselected from the group consisting of SEQ ID NO 1 to SEQ ID NO 10,and/or containing a variant amino acid sequence that is at least 85%homologous to that of SEQ ID NO 1 to SEQ ID NO 10, and/or apolynucleotide containing a nucleic acid encoding SEQ ID NO 1 to SEQ IDNO 10 or the variant amino acid sequence, and a pharmaceuticallyacceptable carrier. Pharmaceutical compositions of the present inventionmay also further comprise at least one additional peptide containing anamino acid sequence selected from the group consisting of SEQ ID NO: 11to SEQ ID NO: 22, or containing a variant amino acid sequence that is atleast 85% identical to that of SEQ ID NO: 11 to SEQ ID NO: 22, orpolynucleotide containing a nucleic acid encoding SEQ ID NO: 11 to SEQID NO: 22 or the variant amino acid sequence. The peptides may have anoverall length of from 8 to 100, from 8 to 30, and from 8 to 17 aminoacids. The peptides may also have non-peptide bonds.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Tetramer analysis of microsphere driven proliferation ofCDC2-001 and ASPM-002 specific CD8+ lymphocytes from peripheral blood ofa healthy donor. 1×10⁶ CD8+ enriched PBMCs per well were stimulatedweekly with microspheres coupled to anti-CD28 plus high density tumorantigen A*2402/CDC2-001 (left panel) or anti-CD28 plus high densitytumor antigen A*024/ASPM-002 (right panel). After three stimulations invitro, all cells were stained with antibody CD8 FITC, andfluorescently-labeled tetramers A*2402/CDC2-001 and A*2402/ASPM-002-001.Cells are gated on CD8+ lymphocytes; numbers in the plots representpercentage of cells in the indicated quadrant among CD8+ lymphocytes(multimer-positive cells).

FIG. 2: Relative in vitro binding of IMA-BIR-002 and IMA-MET-005 derived15-mers to the most frequent HLA-DR alleles. The ProImmune REVEAL™technology employs in vitro HLA-DR assembly assays to determine theon-rates for the MHC:peptide complex as one major determinant of thebinding constant of individual peptides. The assay was performed byProImmune (Oxford, UK). At a fixed time point, the amount of intactMHC:peptide complexes is measured and compared with the amount for apass/fail control (relative weak binder). A strong, promiscuous HLA-DRbinder is included as positive control. Values indicate amount ofbinding for the individual peptides and HLA-DR molecules relative to thepass/fail control. As the REVEAL™ technology is limited to 15-mers, twooverlapping 15-mers (position 2-16; 6-20) were tested instead offull-length MET-005.

FIGS. 3 a and 3 b depict the presence of PSMA and Survivin-specificIFNγ-secreting CD4⁺ T-cells in peripheral blood mononuclear cells (PBMC)from different time points of a vaccinated patient which were determinedusing an IFNγ-EliSpot. Time points: pre-vaccination (a) and after thefter 3^(rd) (b), 6^(th) (c), 7^(th) (d), 8^(th) (e), 9^(th) (f), 10^(th)(g), 11^(th) (h) vaccination.

FIG. 4 shows the presence of Survivin-specific IFNγ-, IL-5, IL-10,TNFα-secreting CD4⁺ T-cells in PBMC from three different time points ofa vaccinated patient which were determined via the Intracellularstaining-Assay (ICS). Time points: after 1^(st) (a), 3^(rd) (b), 7^(th)(c) vaccination.

DETAILED DESCRIPTION OF THE INVENTION

As used herein and except as noted otherwise all terms are defined asgiven below. The term “peptide” is used herein to designate a series ofamino acid residues, connected one to the other typically by peptidebonds between the alpha-amino and carbonyl groups of the adjacent aminoacids. The peptides are preferably 9 amino acids in length, but can beas short as 8 amino acids in length, and as long as 10, 11, 12, 13, 14,15, 16, 17 or 18 amino acids in length.

The term “oligopeptide” is used herein to designate a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.The length of the oligopeptide is not critical to the invention, as longas the correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 14 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse.

A T cell “epitope” requires a short peptide that is bound to a class Ior II MHC receptor, forming a ternary complex (MHC class I alpha chain,beta-2-microglobulin, and peptide) that can be recognized by a T cellbearing a matching T-cell receptor binding to the MHC/peptide complexwith appropriate affinity. Peptides binding to MHC class I molecules aretypically 8-14 amino acids in length, and most typically 9 amino acidsin length. T cell epitopes that bind to MHC class II molecules aretypically 12-30 amino acids in length. In the case of peptides that bindto MHC class II molecules, the same peptide and the corresponding T cellepitope may share a common core segment, but differ in the overalllength due to flanking sequences of differing lengths upstream of theamino-terminus of the core sequence and downstream of itscarboxy-terminus, respectively. MHC class II receptors have a more openconformation, peptides bound to MHC class II receptors arecorrespondingly not completely buried in the structure of the MHC classII molecule peptide-binding cleft as they are in the MHC class Imolecule peptide-binding cleft. Surprisingly this is not the case forthe peptide according to SEQ ID NO: 1 as small variations in the lengthof the peptide lead to an extreme decrease of activity (see below).

In humans there, are three different genetic loci that encode MHC classI molecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-A*11 are examples of different MHC class I alleles that can beexpressed from these loci.

There are three different loci in the human genome for MHC class IIgenes: HLA-DR, HLA-DQ, and HLA-DP. MHC class II receptors areheterodimers consisting of an alpha and a beta chain, both anchoring inthe cell membrane via a transmembrane region. HLA-DRB1*04, andHLA-DRB1*07 are two examples of different MHC class II beta alleles thatare known to be encoded in these loci. Class II alleles are verypolymorphic, e.g. several hundred different HLA-DRB1 alleles have beendescribed. For HLA-A*02 and most frequent HLA-DR serotypes, expressionfrequencies in different populations are shown in Table 2.

TABLE 2 Provides expression frequencies F of HLA*A02 and the mostfrequent HLA-DR serotypes. Frequencies are deduced from haplotypefrequencies G_(f) within the American population adapted from Mori etal. (Mori et al., 1997) employing the Hardy-Weinberg formula F = 1 − (1− G_(f))². Combinations of A*02 with certain HLA-DR alleles might beenriched or less frequent than expected from their single frequenciesdue to linkage disequilibrium. For details refer to Chanock et al.(Chanock et al., 2004). Expression frequencies of HLA*02 and HLA-DRserotypes within North American subpopulations Caucasian African HLAAllele American American Asian American Latin American A*02 49.1% 34.1%43.2% 48.3% DR1 19.4% 13.2% 6.8% 15.3% DR2 28.2% 29.8% 33.8% 21.2% DR320.6% 24.8% 9.2% 15.2% DR4 30.7% 11.1% 28.6% 36.8% DR5 23.3% 31.1% 30.0%20.0% DR6 26.7% 33.7% 25.1% 31.1% DR7 24.8% 19.2% 13.4% 20.2% DR8 5.7%12.1% 12.7% 18.6% DR9 2.1% 5.8% 18.6% 2.1%

Table 3: provides expression frequencies F of HLA*A024 and theHLA*A02402 serotype. Frequencies are deduced from haplotype frequencesG_(f) within the population adapted from Mori et al. (Mori et al., 1997)employing the Hardy-Weinberg formula F=1−(1−Gf)². For details refer toChanock et al. (Chanock et al., 2004).

TABLE 3 Expression frequencies of HLA*02 and A*2402 serotypes worldwideCalculated phenotype Allele Population from Allele Frequency A*24Philippines 65% A*24 Russia Nenets 61% A*2402 Japan 59% A*24 Malaysi 58%A*2402 Philippines 54% A*24 India 47% A*24 South Korea 40% A*24 SriLanka 37% A*24 China 32% A*2402 India 29% A*24 Australia West 22% A*24USA 22% A*24 Russia Samara 20% A*24 South Amerika 20% A*24 Europa 18%

Therefore, for therapeutic and diagnostic purposes a peptide that bindswith appropriate affinity to several different HLA class II receptors ishighly desirable. A peptide binding to several different HLA class IImolecules is called a promiscuous binder.

As used herein, reference to a DNA sequence includes both singlestranded and double stranded DNA. Thus, the specific sequence, unlessthe context indicates otherwise, refers to the single strand DNA of suchsequence, the duplex of such sequence with its complement (doublestranded DNA) and the complement of such sequence. The term “codingregion” refers to that portion of a gene which either naturally ornormally codes for the expression product of that gene in its naturalgenomic environment, i.e., the region coding in vivo for the nativeexpression product of the gene.

The coding region can be from a normal, mutated or altered gene, or caneven be from a DNA sequence, or gene, wholly synthesized in thelaboratory using methods well known to those of skill in the art of DNAsynthesis.

The term “nucleotide sequence” refers to a heteropolymer ofdeoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment,” when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnontranslated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, the claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly contemplated.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form.” As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform.

The term “active fragment” means a fragment that generates an immuneresponse (i.e., has immunogenic activity) when administered, alone oroptionally with a suitable adjuvant, to an animal, such as a mammal, forexample, a rabbit or a mouse, and also including a human, such immuneresponse taking the form of stimulating a T-cell response within therecipient animal, such as a human. Alternatively, the “active fragment”may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion,” “segment,” and “fragment,” whenused in relation to polypeptides, refer to a continuous sequence ofresidues, such as amino acid residues, which sequence forms a subset ofa larger sequence. For example, if a polypeptide were subjected totreatment with any of the common endopeptidases, such as trypsin orchymotrypsin, the oligopeptides resulting from such treatment wouldrepresent portions, segments or fragments of the starting polypeptide.This means that any such fragment will necessarily contain as part ofits amino acid sequence a segment, fragment or portion, that issubstantially identical, if not exactly identical, to a sequence of SEQID NO: 1 to 20, which correspond to the naturally occurring, or “parent”proteins of the SEQ ID NO: 1 to 20. When used in relation topolynucleotides, such terms refer to the products produced by treatmentof said polynucleotides with any of the common endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical,” when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The Percent Identity isthen determined according to the following formula:Percent Identity=100[I−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein

(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and

(ii) each gap in the Reference Sequence and

(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference;

and R is the number of bases or amino acids in the Reference Sequenceover the length of the alignment with the Compared Sequence with any gapcreated in the Reference Sequence also being counted as a base or aminoacid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated Percent Identity is less than the specified PercentIdentity.

The original peptides disclosed herein can be modified by thesubstitution of one or more residues at different, possibly selective,sites within the peptide chain, if not otherwise stated. Suchsubstitutions may be of a conservative nature, for example, where oneamino acid is replaced by an amino acid of similar structure andcharacteristics, such as where a hydrophobic amino acid is replaced byanother hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often shown in correlation with similarities insize, charge, polarity, and hydrophobicity between the original aminoacid and its replacement, and such is the basis for defining“conservative substitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (H, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,amino acids possessing non-standard R groups (i.e., R groups other thanthose found in the common 20 amino acids of natural proteins) may alsobe used for substitution purposes to produce immunogens and immunogenicpolypeptides according to the present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would simultaneously be substituted.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted CTLs, effector functions may be lysisof peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation. For MHC class II-restricted T helper cells,effector functions may be peptide induced secretion of cytokines,preferably, IFN-gamma, TNF-alpha, IL-4, IL5, IL-10, or IL-2, orpeptide-induced degranulation. Possible effector functions for CTLs andT helper cells are not limited to this list.

Immunotherapeutic Approaches for Treatment

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has now raised thepossibility of using a host's immune system to intervene in tumorgrowth. Various mechanisms of harnessing both the humoral and cellulararms of the immune system are currently explored for cancerimmunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofcytotoxic T-cells (CTL) from tumor-infiltrating cell populations or fromperipheral blood suggests that such cells play an important role innatural immune defenses against cancer. CD8-positive T-cells inparticular, which recognize class I molecules of the majorhistocompatibility complex (MHC)-bearing peptides of usually 8 to 10residues derived from proteins or defect ribosomal products (DRIPS)(Schubert U, Antón L C, Gibbs J, Norbury C C, Yewdell J W, Bennink J R.;Rapid degradation of a large fraction of newly synthesized proteins byproteasomes; Nature 2000; 404(6779):770-774) located in the cytosols,play an important role in this response. The MHC-molecules of the humanare also designated as human leukocyte-antigens (HLA).

There are two classes of MHC-molecules: MHC class I molecules that canbe found on most cells having a nucleus which present peptides thatresult from proteolytic cleavage of mainly endogenous, cytosolic ornuclear proteins, DRIPS, and larger peptides. However, peptides derivedfrom endosomal compartments or exogenous sources are also frequentlyfound on MHC class I molecules. This non-classical way of class Ipresentation is referred to as cross-presentation in literature. MHCclass II molecules can be found predominantly on professional antigenpresenting cells (APCs), and present predominantly peptides of exogenousproteins that are taken up by APCs during the course of endocytosis, andare subsequently processed. As for class I, alternative ways of antigenprocessing are described that allow peptides from endogenous sources tobe presented by MHC class II molecules (e.g. autophagocytosis).Complexes of peptide and MHC class I molecule are recognized byCD8-positive cytotoxic T-lymphocytes bearing the appropriate TCR,whereas complexes of peptide and MHC class II molecule are recognized byCD4-positive helper T-cells bearing the appropriate TCR.

CD4-positive helper T-cells play an important role in orchestrating theeffector functions of anti-tumor T-cell responses and for this reasonthe identification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) may be of great importance for the developmentof pharmaceutical products for triggering anti-tumor immune responses(Gnjatic, S., D. Atanackovic, E. Jäger, M. Matsuo, A. Selvakumar, N. K.Altorki, R. G. Maki, B. Dupont, G. Ritter, Y. T. Chen, A. Knuth, and L.J. Old. Survey of naturally occurring CD4+ T-cell responses againstNY-ESO-1 in cancer patients: Correlation with antibody responses. Proc.Natl. Acad. Sci. U.S.A. 2003, 100 (15): 8862-7) CD4+ T cells can lead tolocally increased levels of IFN-gamma.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CTL effector cells (i.e., CD8-positive T lymphocytes),CD4-positive T-cells are sufficient for inhibiting manifestation oftumors via inhibition of angiogenesis by secretion of interferon-gamma(IFNγ) (Qin, Z. and T. Blankenstein. CD4+T-cell-mediated tumor rejectioninvolves inhibition of angiogenesis that is dependent on IFN gammareceptor expression by nonhematopoietic cells. Immunity. 2000,12:677-686). Additionally, it was shown that CD4-positive T-cellsrecognizing peptides from tumor-associated antigens presented by HLAclass II molecules can counteract tumor progression via the induction ofan antibody (Ab) responses (Kennedy, R. C., M. H. Shearer, A. M. Watts,and R. K. Bright. CD4+T lymphocytes play a critical role in antibodyproduction and tumor immunity against simian virus 40 large tumorantigen. Cancer Res. 2003, 63:1040-1045). In contrast totumor-associated peptides binding to HLA class I molecules, only a smallnumber of class II ligands of TAA have been described so far.

Since the constitutive expression of HLA class II molecules is usuallylimited to cells of the immune system the possibility of isolating classII peptides directly from primary tumors was not considered possible.However, the inventors were recently successful in identifying a numberof MHC class II epitopes directly from tumors (EP 1642905, EP 1760088;Dengjel J, Nastke M D, Gouttefangeas C, Gitsioudis G, Schoor O,Altenberend F, Müller M, Krämer B, Missiou A, Sauter M, Hennenlotter J,Wernet D, Stenzl A, Rammensee H G, Klingel K, Stevanović S.; Unexpectedabundance of HLA class II presented peptides in primary renal cellcarcinomas; Clin Cancer Res. 2006; 12:4163-4170).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially APCs, e.g.,monocytes, monocyte-derived cells, macrophages, dendritic cells. Intumor patients, cells of the tumor have surprisingly been found toexpress MHC class II molecules (Dengjel J, Nastke M D, Gouttefangeas C,Gitsioudis G, Schoor O, Altenberend F, Müller M, Krämer B, Missiou A,Sauter M, Hennenlotter J, Wernet D, Stenzl A, Rammensee H G, Klingel K,Stevanović S.; Unexpected abundance of HLA class II presented peptidesin primary renal cell carcinomas; Clin Cancer Res. 2006; 12:4163-4170).

For a peptide to trigger (elicit) a cellular immune response, it mustbind to an MHC-molecule. This process is dependent on the allele of theMHC-molecule and specific polymorphisms of the amino acid sequence ofthe peptide. MHC-class-I-binding peptides are usually 8-10 amino acidresidues in length and usually contain two conserved residues (“anchor”)in their sequence that interacts with the corresponding binding grooveof the MHC-molecule. In this way each MHC allele has a “binding motif”determining which peptides can bind specifically to the binding groove(Rammensee H G, Bachmann J, Stevanovic S. MHC ligands and peptidemotifs, Landes Bioscience, USA, 1997).

In MHC dependent immune reaction, peptides not only have to be able tobind to certain MHC molecules expressed by tumor cells, they also haveto be recognized by T-cells bearing specific T-cell receptors (TCR).

The antigens that are recognized by the tumor specific T-lymphocytes,that is, their epitopes, can be molecules derived from all proteinclasses, such as enzymes, receptors, transcription factors, etc.Furthermore, tumor-associated antigens, for example, can also be presentin tumor cells only, for example as products of mutated genes. Anotherimportant class of tumor-associated antigens are tissue-specificantigens, such as CT (“cancer testis”)-antigens that are expressed indifferent kinds of tumors and in healthy tissue of the testis.

Various tumor-associated antigens have been identified. Further, muchresearch effort is expended to identify additional tumor associatedantigens. Some groups of tumor-associated antigens, also referred to inthe art as tumor-specific antigens, are tissue specific. Examplesinclude, but are not limited to, tyrosinase for melanoma, PSA and PSMAfor prostate cancer and chromosomal cross-overs (translocations) such asbcr/abl in lymphoma. However, many tumor-associated antigens identifiedoccur in multiple tumor types, and some, such as oncogenic proteinsand/or tumor suppressor genes (tumor suppressor genes are, for examplereviewed for renal cancer in Linehan W M, Walther M M, Zbar B. Thegenetic basis of cancer of the kidney. J. Urol. 2003 December; 170(6Pt1):2163-72) which actually cause the transformation event, occur innearly all tumor types. For example, normal cellular proteins thatcontrol cell growth and differentiation, such as p53 (which is anexample for a tumor suppressor gene), ras, c-met, myc, pRB, VHL, andHER-2/neu, can accumulate mutations resulting in upregulation ofexpression of these gene products thereby making them oncogenic{McCartey1998} (McCartey et al. Cancer Research, 1998, 15:58 2601-5;Disis et al. Ciba Found. Symp. 1994, 187:198-211). These mutant proteinscan also be a target of a tumor-specific immune response in multipletypes of cancer.

Immunotherapy in cancer patients aims at activating cells of the immunesystem specifically, especially the so-called cytotoxic T-cells (CTL,also known as “killer cells”, also known as CD8-positive T-cells),against tumor cells but not against healthy tissue. Tumor cells differfrom healthy cells by the expression of tumor-associated proteins. HLAmolecules on the cell surface present the cellular content to theoutside, thus enabling a cytotoxic T cell to differentiate between ahealthy and a tumor cell. This is realized by breaking down all proteinsinside the cell into short peptides, which are then attached to HLAmolecules and presented on the cell surface (Rammensee et al., 1993).Peptides that are presented on tumor cells, but not or to a far lesserextent on healthy cells of the body, are called tumor-associatedpeptides (TUMAPs).

For proteins to be recognized by cytotoxic T-lymphocytes astumor-specific or -associated antigens, and to be used in a therapy,particular prerequisites must be fulfilled. The antigen should beexpressed mainly by tumor cells and not by normal healthy tissues or incomparably small amounts. It is furthermore desirable, that therespective antigen is not only present in a type of tumor, but also inhigh concentrations (i.e. copy numbers of the respective peptide percell). Tumor-specific and tumor-associated antigens are often derivedfrom proteins directly involved in transformation of a normal cell to atumor cell due to a function e.g. in cell cycle control or apoptosis.Additionally, downstream targets of the proteins directly causative fora transformation may be upregulated and thus may be indirectlytumor-associated. Such indirect tumor-associated antigens may also betargets of a vaccination approach. In both cases the presence ofepitopes in the amino acid sequence of the antigen is essential, sincesuch peptide (“immunogenic peptide”) that is derived from a tumorassociated antigen should lead to an in vitro or in vivoT-cell-response.

Basically, any peptide able to bind a MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T-cell with a correspondingTCR and the absence of tolerance for this particular epitope. T-helpercells play an important role in orchestrating the effector function ofCTLs in anti-tumor immunity. T-helper cell epitopes that trigger aT-helper cell response of the TH1 type support effector functions ofCD8-positive killer T-cells, which include cytotoxic functions directedagainst tumor cells displaying tumor-associated peptide/MHC complexes ontheir cell surfaces. In this way tumor-associated T-helper cell peptideepitopes, alone or in combination with other tumor-associated peptides,can serve as active pharmaceutical ingredients of vaccine compositionsthat stimulate anti-tumor immune responses.

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+CTLs (MHC class I molecule) or by CD4-positive CTLs (MHC class IImolecule) is important in the development of tumor vaccines. It istherefore an object of the present invention, to provide compositions ofpeptides that contain peptides binding to MHC complexes of either class.

First clinical trials using tumor-associated peptides started in themid-1990s by Boon and colleagues mainly for melanoma. Clinical responsesin the best trials have ranged from 10% to 30%. Severe side effects orsevere autoimmunity have not been reported in any clinical trial usingpeptide-based vaccine monotherapy. Mild forms of vitiligo have beenreported for some patients who had been treated with melanoma-associatedpeptides.

However, priming of one kind of CTL is usually insufficient to eliminateall tumor cells. Tumors are very mutagenic and thus able to respondrapidly to CTL attacks by changing their protein pattern to evaderecognition by CTLs. To counter-attack the tumor evasion mechanisms avariety of specific peptides is used for vaccination. In this way abroad simultaneous attack can be mounted against the tumor by severalCTL clones simultaneously. This may decrease the chances of the tumor toevade the immune response. This hypothesis has been recently confirmedin a clinical study treating late-stage melanoma patients. With only fewexceptions, patients that had at least three distinct T-cell responses,showed objective clinical responses or stable disease (Banchereau etal., 2001) as well as increased survival (personal communication with J.Banchereau), while the vast majority of patients with less than threeT-cell responses were diagnosed with progressive disease.

A study of the applicants showed a similar effect when patientssuffering from renal cell carcinoma were treated with a vaccine composedof 13 different peptides (H. Singh-Jasuja, S. Walter, T. Weinschenk, A.Mayer, P. Y. Dietrich, M. Staehler, A. Stenzl, S. Stevanovic, H.Rammensee, J. Frisch; Correlation of T-cell response, clinical activityand regulatory T-cell levels in renal cell carcinoma patients treatedwith IMA901, a novel multi-peptide vaccine; ASCO Meeting 2007 Poster#3017; M. Staehler, A. Stenzl, P. Y. Dietrich, T. Eisen, A. Haferkamp,J. Beck, A. Mayer, S. Walter, H. Singh, J. Frisch, C. G. Stief; An openlabel study to evaluate the safety and immunogenicity of the peptidebased cancer vaccine IMA901, ASCO meeting 2007; Poster #3017).

The major task in the development of a tumor vaccine is therefore notonly the identification and characterization of novel tumor associatedantigens and immunogenic T-helper epitopes derived thereof, but also thecombination of different epitopes to increase the likelihood of aresponse to more than one epitope for each patient. It is therefore anobject of the present invention to provide combinations of amino acidsequences of such peptides that have the ability to bind to a moleculeof the human major histocompatibility complex (MHC) class-I (HLA classI) or II (HLA class II). It is a further object of the presentinvention, to provide an effective anti-cancer vaccine that is based ona combination of the peptides.

In the present invention, the inventors isolated and characterizedpeptides binding to HLA class I or II molecules directly from mammaliantumors, i.e. primary samples of mainly gastric cancer patients, but alsofrom primary tissue samples of glioblastoma, colorectal cancers, renalcell carcinoma, lung cancers, pancreatic cancers, malignant melanoma,and cancer of the stomach.

The present invention provides peptides that stem from antigensassociated with tumorigenesis, and have the ability to bind sufficientlyto MHC(HLA) class II molecules for triggering an immune response ofhuman leukocytes, especially lymphocytes, especially T lymphocytes,especially CD4-positive T lymphocytes, especially CD4-positive Tlymphocytes mediating T_(H1)-type immune responses.

The present invention also provides peptides that stem from antigensassociated with tumorigenesis, and have the ability to bind sufficientlyto MHC(HLA) class I molecules for triggering an immune response of humanleukocytes, especially lymphocytes, especially T lymphocytes, especiallyCD8-positive cytotoxic T-lymphocytes as well as combinations of the twothat are particularly useful for vaccination of patients that sufferfrom cancer.

According to the present invention, the object is solved by providing apharmaceutical composition comprising at least two peptides containingan amino acid sequence selected from the group consisting of SEQ ID NO 1to SEQ ID NO 10, and/or containing a variant amino acid sequence that isat least 85% homologous to that of SEQ ID NO 1 to SEQ ID NO 10, and/or apolynucleotide containing a nucleic acid encoding SEQ ID NO 1 to SEQ IDNO 10 or the variant amino acid sequence, and a pharmaceuticallyacceptable carrier. Pharmaceutical compositions of the present inventionmay also further comprise at least one additional peptide containing anamino acid sequence selected from the group consisting of SEQ ID NO: 11to SEQ ID NO: 22, or containing a variant amino acid sequence that is atleast 85% identical to that of SEQ ID NO: 11 to SEQ ID NO: 22, orpolynucleotide containing a nucleic acid encoding SEQ ID NO: 11 to SEQID NO: 22 or the variant amino acid sequence. The peptides may have anoverall length of not more than 100, preferably not more than 30, andmost preferably from 8 to 17 amino acids. The peptides may also havenon-peptide bonds.

As described herein below, the peptides that form the basis of thepresent invention have all been identified as presented by MHC class Ior II bearing cells. Thus, these particular peptides as well as otherpeptides containing the sequence (i.e. derived peptides) all elicit aspecific T-cell response, although the extent to which such responsewill be induced might vary from individual peptide to peptide and fromindividual patient to patient. Differences, for example, could be causeddue to mutations in the peptides. The person of skill in the present artis well aware of methods that can be applied to determine the extent towhich a response is induced by an individual peptide, in particular withreference to the examples herein and the respective literature.

Preferably the variants of the invention will induce T-cellscross-reacting with the respective peptide of the invention.

The peptides stem from tumor-associated antigens, especiallytumor-associated antigens with functions in, e.g., proteolysis,angiogenesis, cell growth, cell cycle regulation, cell division,regulation of transcription, regulation of translation, tissue invasion,etc. Table 4 provides the peptides and the function of the protein thepeptides are derived from.

TABLE 4 Peptides of the present invention and function of the parent protein SEQ ID Gene  binds to NO Peptide IDSequence Symbol MHC 1 CDC2-001 LYQILQGIVF CDK1 HLA-A*024 2 ASPM-002SYNPLWLRI ASPM HLA-A*024 3 UCHL5-001 NYLPFIMEL UCHL5 HLA-A*024 4 MET-006SYIDVLPEF MET HLA-A*024 5 PROM1-001 SYIIDPLNL PROM1 HLA-A*024 6UQCRB-001 YYNAAGFNKL UQCRB HLA-A*024 7 MST1R-001 NYLLYVSNF MST1RHLA-A*024 8 PPAP2C-001 AYLVYTDRL PPAP2C HLA-A*024 9 SMC4-001 HYKPTPLYFSMC4 HLA-A*024 10 MMP11-001 VWSDVTPLTF MMP11 HLA-A*024 20 AVL9-001FYISPVNKL AVL9 HLA-A*024 24 ERBB3-001 VYIEKNDKL ERBB3 HLA-A*024

TABLE 5 Additional immunogenic peptides useful in a composition of the invention binds to SEQ ID NO Peptide ID SequenceGene Symbol MHC 11 BIR-002 TLGEFLKLDRERAKN BIRC5 HLA-DR and HLA- A*02 12CDC42- DDPSTIEKLAKNKQKP CDC42 HLA-DR 001 13 CDC42- NKQKPITPETAEKLARDCDC42 HLA-DR 002 14 SPP1-001 NGAYKAIPVAQDLNAPS SPP1 HLA-DR 15 BIR-002aTLGEFLKLDRERAKD Survivin HLA-DR and HLA- A*02 16 BIR-002b FTELTLGEFSurvivin HLA-A1 17 BIR-002c LMLGEFLKL Survivin HLA-A2 18 BIR-002dEPDLAQCFY Survivin HLA-B35 19 NUF2-001 VYGIRLEHF NUF2 HLA- A*024 20AVL9-001 FYISPVNKL AVL9 HLA- A*024 21 ABL1-001 TYGNLLDYL ABL1 HLA- A*02422 NUF2-002 RFLSGIINF NUF2 HLA- A*024 23 (HBV-001) FLPSDFFPSV controlpeptideCell Division Cycle 2 Protein (CDC2)

CDC2, also known as p34cdc2 or Cdk1 (Cyclin-dependent kinase 1), belongsto the Cdks, a family of serine/threonine protein kinases, and plays akey role in cell cycle control. It is known as the main regulator of theG2-to-M transition. At the end of interphase, it is activated by bindingto A-type cyclins and facilitates the onset of mitosis. After breakdownof the nuclear envelope, A-type cyclins are degraded and replaced bycyclin B. The complex between CDC2 and cyclinB forms the mitosispromoting factor (MPF), which is essential for driving cells throughmitosis.

Active CDC2 phosphorylates more than 70 substrates. For example,phosphorylation of the “linker” histone H1 leads to a relaxation ofchromatin structure and transcription of specific genes, andphosphorylation of RNA-polymerase II enhances transcription (Cisek andCorden, 1989). BRCA2 phosphorylation stimulates homologousrecombination-dependent repair (Esashi et al., 2005), and FOXO1phosphorylation inhibits its transcriptional activity, resulting in cellproliferation and survival. Phosphorylation of separase inhibitspremature sister chromatid separation (Stemmann et al., 2001). CDC2activity is switched off again during anaphase, as theanaphase-promoting complex/cyclosome (APC/C) ubiquitinates cyclin B,leading to its degradation.

The function of CDC2 in mitosis is non-redundant and cannot becompensated by the activity of other Cdks such as Cdk2, 4 and 6. Bycontrast, CDC2 was reported to function in other phases of the cellcycle such as the G1-S transition as well, and it is able to substitutefor the “interphase Cdks”. Thus, CDC2 was proposed to be the onlyessential cell cycle Cdk.

Apart from its expression and function in cell cycle, it was reportedthat in some cases CDC2 is expressed in apoptotic conditions, and thatenhanced activity can lead to mitotic catastrophe. Overexpression ofCDC2 was found in several cancers, although the expression of other cellcycle proteins such as cyclins is dysregulated even more frequently.Among the cancer types overexpressing CDC2 are prostate carcinoma, oralcavity carcinomas, oral squamous carcinoma (OSCC), acute myeloidleukemia (AML) (Qian et al., 2009), H. pylori-induced MALT lymphoma(Banerjee et al., 2000) and colon carcinoma (Yasui et al., 1993). Inseveral cases, overexpression was correlated with poor prognosis. Ingastric carcinoma (GC), overexpression and/or enhanced activity has beenreported (14 of 23 cases), and it was suggested that CDC2 overexpressioncould play a causative role. Furthermore, CDC2 was found to be among aset of genes active during mitosis, which, if overexpressed, lead tochromosomal instability of tumors. Inhibitors of CDC2 and other Cdkshave been considered as drug candidates for cancer therapy (Shapiro,2006).

Abnormal Spindle-Like Microcephaly Associated Protein (ASPM)

Abnormal spindle-like microcephaly associated (ASPM) gene is the humanorthologue of the Drosophila abnormal spindle (asp) and the mostcommonly mutated gene of autosomal recessive primary microcephaly. Inhuman, the defective neurogenesis caused by homozygous mutation of ASPMleads to microcephaly and mental retardation.

The most common cause of primary autosomal recessive microcephaly (MCPH)appears to be mutations in the ASPM gene which is involved in theregulation of neurogenesis. ASPM is localized in the spindle polesduring mitosis.

ASPM inhibition by siRNA-mediated knockdown inhibits tumor cellproliferation and neural stem cell proliferation, supporting ASPM as apotential molecular target in glioblastoma. ASPM was overexpressed inglioblastoma relative to normal brain. The expression of ASPM may beused as a marker for glioma malignancy and represents a potentialtherapeutic target. ASPM overexpression is a molecular marker predictingenhanced invasive/metastatic potential of hepatocellular carcinoma HCC,higher risk of early tumor recurrence ETR regardless of p53 mutationstatus and tumor stage, and hence poor prognosis. ASPM was alsoupregulated in immortalized cells, cancer cells, and non-small-cell lungcancer (NSCLC) tissues (Jung et al., 2009).

Ubiquitin Carboxyl-Terminal Hydrolase L5 (UCHL5)

Ubiquitin carboxyl-terminal hydrolase L5 (UCHL5), also known asUbiquitin C-terminal hydrolase (UCH37) or INO80R, is a deubiquitinasethat is associated with the proteasome. It disassembles protein-attachedpolyubiquitin chains from the distal end by cleaving the isopeptide bondbetween the C-terminal Cys76 and Lys48 (Nishio et al., 2009).

UCHL5 binds to hRpn13 (also known as Adrm1), a component of the 19S(PA700) regulatory particle of the proteasome, which activates itsisopeptidase activity. hRpn13 also functions as receptor for ubiquitin,thereby coupling substrate recognition to deubiquitination. UCHL5 canalso bind to Rpn10/S5a, another component of the 19S particle locatedbetween its “lid” and its “base”, but this interaction fails to activateUCHL5. UCH37 could rescue poorly ubiquitinated substrates or otherslowly degraded Ub-conjugates from proteolysis. Alternatively, it mayalso enhance degradation by facilitating release of polyubiquitinatedsubstrates from their initial binding site in the 19S regulatory complexfor translocation into the proteolytic core, the 20S particle of the 26Sproteasome. In the nucleus, UCHL5 is also associated with the Ino80chromatin-remodeling complex. Upon binding of a proteasome, it becomesactivated and may contribute to the regulation of transcription or DNArepair that has been suggested to be mediated by Ino80 and theproteasome. The function of UCHL5 might at least partly be exerted byother proteins, as RNAi-mediated knockdown of UCHL5 had no detectableeffect on cell growth, proteasome structure or proteolytic capacity,although it accelerated cellular protein degradation.

Ubiquitin specific proteases like UCHL5 are involved in severalprocesses such as control of cell cycle progression, differentiation,DNA replication and repair, transcription, protein quality control,immune response and apoptosis. There is at least some evidence thatUCHL5 contributes to malignant transformation. Its activity has beenshown to be upregulated in human cervical carcinoma tissue as comparedto adjacent normal tissue. It is able to deubiquitinate and therebystabilize the TGF-beta receptor and its downstream mediators, the Smads,thereby enhancing TGF-beta signaling. Enhanced TGF-beta signaling canact as a tumor promoter in late stages of cancer progression, althoughit has a dual function and can also be a tumor suppressor in earlystages and before initiation (Wicks et al., 2005; Wicks et al., 2006;Horton et al., 2007; Bierie and Moses, 2006).

c-Met

See for example EP 08008292.8 and EP1507795B1. Furthermore, constitutivec-Met activation through phosphorylation has also been identified as animportant mechanism of oncogenesis in human clear-cell renal cellcarcinoma (Nakaigawa et al., 2006).

MET over-expression, often induced by tumor hypoxia, leads toconstitutive activation of the receptor and correlates with poorprognosis. Silencing the endogenous MET gene, over-expressed in tumorcells, results in impairment of the execution of the full invasivegrowth program in vitro, lack of tumor growth and decreased generationof experimental metastases in vivo. Notably, silencing MET in alreadyestablished metastases leads to their almost complete regression (Corsoet al., 2008).

Macrophage-Stimulating Protein Receptor (MST1R)

The MST1R (alias RON) receptor is a member of the Met family of cellsurface receptor tyrosine kinases and is primarily expressed onepithelial cells and macrophages. Like c-MET, RON is expressed by avariety of epithelial-derived tumors and cancer cell lines and it isthought to play a functional role in tumorigenesis. Clinical studieshave shown that MST1R overexpression is associated with both worsepatient outcomes as well as metastasis.

MST1R expression is significant in gastric carcinoma tissue andcorresponding paraneoplastic tissue, but is not expressed in normalgastric mucosa (Zhou et al., 2008). MST1R receptor can induce cellmigration, invasion, proliferation and survival in response to itsrespective ligand. Moreover, MST1R possess oncogenic activity in vitro,in animal models in vivo and is often deregulated in human cancers(Dussault and Bellon, 2009). Data show that knockdown of MST1R inprostate cancer cells results in significantly less endothelial cellchemotaxis when compared with MST1R-expressing cells in vitro as well asin reduced tumor growth and decreased microvessel density afterorthotopic transplantation into the prostate in vivo. It has been shownthat RNA interference-mediated knockdown of MST1R kinase in a highlytumorigenic colon cancer cell line led to reduced proliferation ascompared with the control cells.

Structural Maintenance of Chromosomes Proteins 4 (SMC4)

Structural maintenance of chromosomes (SMC) proteins are chromosomalATPases, highly conserved from bacteria to humans, that play fundamentalroles in many aspects of higher-order chromosome organization anddynamics.

The SMC4 protein is a core component of the condensin complex that playsa role in chromatin condensation and has also been associated withnucleolar segregation, DNA repair, and maintenance of the chromatinscaffold. Eukaryotes have at least six SMC proteins in individualorganisms, and they form three distinct heterodimers with specializedfunctions: SMC2 and SMC4 function as the core of the condensin complexesthat are essential for chromosome assembly and segregation.

Analysis of mRNA levels in 25 different normal tissues by RT-PCR showsthat this gene is expressed highly in normal prostate and salivarygland, very weakly in colon, pancreas, and intestine, and not at all inother tissues. RT-PCR studies on human cancer samples show that the RNAis expressed highly in many cancer cell lines and cancer specimens,including 26 of 33 human breast cancers, 3 of 3 prostate cancers, 3 of 3colon cancers, and 3 of 3 pancreatic cancers (Egland et al., 2006).

AVL9

Surprisingly, this protein was found as source protein, since only poordata is available about the AVL9 protein and the function of thecorresponding gene.

Kinetochore Protein Nuf2

NUF2 (CDCA-1) gene encodes a protein that is highly similar to yeastNuf2, a component of a conserved protein complex associated with thecentromere. Yeast Nuf2 disappears from the centromere during meioticprophase when centromeres lose their connection to the spindle polebody, and plays a regulatory role in chromosome segregation. It wasshown that survivin and hNuf2 csiRNAs temporally knockdown their mRNAscausing multinucleation and cell death by mitotic arrest, respectively(Nguyen et al., 2006). Nuf2 and Hec1 are required for organization ofstable microtubule plus-end binding sites in the outer plate that areneeded for the sustained poleward forces required for biorientation atkinetochores (DeLuca et al., 2005).

Immunohistochemical analysis using lung cancer tissue microarrayconfirmed high levels of CDCA1 and KNTC2 proteins in the great majorityof lung cancers of various histologic types. Their elevated expressionswere associated with poorer prognosis of NSCLC patients. Inhibition oftheir binding by a cell-permeable peptide carrying the CDCA1-derived19-amino-acid peptide (11R-CDCA1(398-416)) that correspond to thebinding domain to KNTC2 effectively suppressed growth of NSCLC cells(Hayama et al., 2006). siRNA-mediated knockdown against CDCA1 or KNTC2has been found to inhibit cell proliferation and induction of apoptosisin NSCLC, ovarian cancer, cervical cancer, gastric cancer, colorectalcancer and glioma (Kaneko et al., 2009). CDCA 1 gene is differentiallyexpressed in cervical cancer (expression of mRNA validated by real-timePCR and protein by immunohistochemistry) (Martin et al., 2009). RT-PCRwith surgically resected gastric cancer tissues (diffuse type, 6;intestinal type, 4) confirmed that two variants of CDCA1 wereupregulated in cancer tissues. Alternative splicing variants, especiallyin CDCA1, were detected in this study and may be potentially useful asdiagnostic markers and/or novel targets for anticancer therapy (Ohnumaet al., 2009).

Lipid Phosphate Phosphohydrolase 2 (PPAP2C)

The protein encoded by this gene is a member of the phosphatidic acidphosphatase (PAP) family. PAPs convert phosphatidic acid todiacylglycerol, and function in de novo synthesis of glycerolipids aswell as in receptor-activated signal transduction mediated byphospholipase D. Three alternatively spliced transcript variantsencoding distinct isoforms have been reported.

PPAP2C is a potentially novel target that is up-regulated in transformedprimary human adult mesenchymal stem cells MSC. Knockdown of PPAP2Cdecreases cell proliferation by delaying entry into S phase of the cellcycle and is transcriptionally regulated by p53. Some data suggest thatoverexpression of PPAP2C, observed in numerous human cancers, may be arequirement for increased cell proliferation (Flanagan et al., 2009). Astudy demonstrates that PPAP2C is a regulator of cell cycle progressionin fibroblasts. Overexpression of PPAP2C, but not a catalyticallyinactive mutant, caused premature S-phase entry, accompanied bypremature cyclin A accumulation. These represent substantial changes inthe rate of S-phase entry that could have implications in processes suchas mitogenesis, migration, wound healing, development, and tumorgenesis.

Ubiquinol-Cytochrome c Reductase Binding Protein (UQCRB)

The UQCRB-gene encodes a protein that is part of theubiquinol-cytochrome c oxidoreductase complex which contains tennuclear-encoded and one mitochondrial-encoded subunits. The encodedprotein binds ubiquinone and participates in the transfer of electronswhen ubiquinone is bound. Mutations in this gene are associated withmitochondrial complex III deficiency. A pseudogene has been described onthe X chromosome.

The UQCRB-gene may be a potential oncogene or a tumour suppressor genein pancreatic ductal adenocarcinoma (Harada et al., 2009). TheUQCRB-gene is overexpressed in hepatocellular carcinoma (Jia et al.,2007).

Prominin 1 (Prom1)

Prominin-1, also called CD133, was originally identified as a moleculespecific for CD34+ hematopoetic progenitor cells and later on shown tobe a marker for normal stem cells and cancer stem cells (CSCs) ofvarious tissues (Mizrak et al., 2008). However, little is known aboutits function. As it is located mainly in protrusions of the plasmamembrane, such as the microvilli of epithelial cells, a functional rolewas ascribed to prominin-1 as an ‘organizer’ of plasma membranetopology. As it was found to interact with cholesterol, it might beimportant in maintaining an appropriate lipid composition within theplasma membrane.

Prominin-1 is used as CSC marker in many human tumors. Only a smallpercentage of tumor cells is usually positive for prominin-1, asexpected for a CSC marker. Depending on the tumor type, the number ofpositive cells per tumor mass reaches from 1 to 15% and is mostly around2%. Tumors where prominin-1 expressing cells have been shown to be CSCsby functional tests (such as sphere formation, high capacity to initiateof tumor growth in immunodeficient mice and asymmetricdivision/self-renewal/pluripotency) are:

-   -   colon cancer (2-2.5% of tumor mass) (Todaro et al., 2007;        Ricci-Vitiani et al., 2007),    -   liver cancer (Ma et al., 2007; Suetsugu et al., 2006; Yin et        al., 2007),    -   pancreatic cancer (Hermann 2007; Wang 2009),    -   prostate cancer (1% of tumor mass) (Richardson et al., 2004),    -   brain tumors of different phenotypes (Singh et al., 2003; Singh        et al., 2004),    -   leukemias such as acute lymphoblastic leukemia, ALL (Cox et al.,        2009),    -   melanoma (Monzani et al., 2007; Rappa et al., 2008),    -   lung cancer (Chen and O'Shea, 2008; Eramo et al., 2008; Tirino        et al., 2009),    -   Ewing's sarcoma (Suva et al., 2009),    -   endometrial cancer (Rutella et al., 2009),    -   oral squamous cell carcinoma (Zhang et al., 2009) and    -   head and neck squamous cell carcinoma (Harper et al., 2007).

Moreover, several studies show an increased prominin-1 expression incancerous tissue as compared to healthy tissue, and most of them find acorrelation of prominin-1 expression with clinical parameters such asoverall survival, tumor stage or metastasis. Examples are non-small celllung cancer, malignant melanoma, retinoblastoma, neuroblastoma andsynovial carcinoma. Prominin-1 expression also correlated with poorprognosis in glioma, pancreatic cancer (up to 15% PROM1+ cells),colorectal, rectal and colon cancer and ductal breast carcinoma.Interestingly, PROM1 mRNA is upregulated in PBMCs of cancer patientswith metastatic disease, especially in patients with bone metastasis,and PROM1 expression in PBMCs is a prognostic factor for overallsurvival. No correlation with prognosis was found in ovarian cancer. Indiffuse gastric cancer, PROM1 expression was suggested based on an insilico analysis (Katoh and Katoh, 2007) and overexpression in gastriccancer compared to normal stomach tissue at the protein level wasreported by (Smith et al., 2008). However, (Boegl and Prinz, 2009)reported that prominin-1 expression was reduced in gastric cancer,especially in later stages, and claimed that prominin-1 expressionrather correlates with angiogenesis—which is also reduced in laterstages—than with tumor growth. A study using gastric cancer cell lines(Takaishi et al., 2009) claims that CD44, but not prominin-1 is a CSCmarker in gastric cancer.

Evidence for involvement of prominin-1 expressing cells in tumorformation was provided by (Zhu et al., 2009), who reported that in amouse intestinal cancer model, all neoplastic cells arose from Prom1+cells, but only 7% retained the Prom1+ phenotype. Apart from that,prominin-1(+) cells have been shown to contribute to tumor angiogenesis.As expected for CSCs, prominin-1(+) cells have been shown to bechemoresistant due to activation of the Akt survival pathway (Ma 2008).(Bertolini et al., 2009) report that they do not respond to cisplatintreatment. They are resistant to TRAIL- and Fas-induced apoptosis due toupregulation of FLIP. They protect themselves from apoptosis bysecretion of IL-4. However, they might be accessible by the immunesystem, as they can be killed by NK cells (Castriconi et al., 2007;Pietra et al., 2009) and cytotoxic T cells (Brown et al., 2009).

Matrix Metalloproteinase 11 (MMP11)

Matrix metalloproteinase 11 (MMP11) was proposed to play a role duringseveral physiological processes requiring tissue remodeling, such asdevelopment, postlactating involution of the mammary gland, woundhealing and scar formation and during the menstrual cycle. It has alsobeen proposed to negatively regulate fat homeostasis by reducingadipocyte differentiation. In contrast to other MMPs, it is not able tocleave typical extracellular matrix molecules—except collagen VI.However, other substrates have been identified such as alpha2-macroglobulin, certain serine protease inhibitors (serpins) includingalpha 1 anti-trypsin, insulin-like growth factor-binding protein-1 andthe laminin receptor.

MMP11 was discovered as a gene that is overexpressed specifically instromal cells surrounding invasive breast carcinoma. Further studiesconfirmed its expression in the tumor-surrounding stroma of breastcarcinoma and of other cancer types, such as skin cancer, non-small cellas well as small cell lung carcinomas, head and neck squamous cellcarcinoma, colon and colorectal carcinoma, epithelial cancer of thelarynx, oesophageal carcinoma, oral carcinoma, pancreatic carcinoma,carcinoma of the urinary bladder, ovarian carcinomas, renal cellcarcinoma, atypical meningioma, papillary thyroid carcinoma, braintumors (MMP11 was expressed in astrocytomas, but only to a low extent inoligodendrogliomas, and in malignant gliomas), salivary duct carcinoma,cervical cancer, extranodal T/NK-cell lymphomas, Non-Hodgkins Lymphomaand prostate carcinoma. It was stated that MMP11 is overexpressed in thestroma of most invasive human carcinomas, but rarely in sarcomas andother nonepithelial tumors Mostly, MMP11 is expressed in stroma cellsdirectly adjacent to the tumor, whereas the tumor cells themselves,normal tissues and stroma cells distant from the tumor are negative.However, this cannot be generalized as in some cases MMP11 was alsofound in noncancerous tissue such as that of the colon or in tumorcells, e.g. in tumors of the pancreas, breast, arachnoid membrane andstomach. Higher levels of MMP11 are correlated with a malignantphenotype/higher invasiveness and bad prognosis. However, in papillarythyroid carcinomas, MMP11 expression was inversely linked to aggressivecharacteristics.

A role in angiogenesis is not probable, as MMP11 expression did notcorrelate with microvessel density. Rather, it appears to enhance cancercell survival and suppress apoptosis. It was proposed that MMP11 fromfibroblasts leads to the stimulation of the IGF-1R pathway in carcinomacells, thus enhancing their proliferative capacity. Its capacity to leadto adipocyte dedifferentiation supports cancer by accumulation ofperitumoral fibroblast-like cells which favor cancer cell survival andtumor progression (Motrescu and Rio, 2008). MMP11 was found in tumortissue as well as in serum of gastric cancer patients, and expressioncorrelated with metastasis (Yang et al., 2008). Moreover, (Deng et al.,2005) showed that MMP11 is highly expressed in tumor cell lines andprimary tumor of gastric cancer—in contrast to other cancer types notexclusively in the stroma—and that it appears to enhance tumor cellproliferation.

ABL1

The ABL1 protooncogene encodes a cytoplasmic and nuclear proteintyrosine kinase of the Src family that has been implicated in processesof cell differentiation, cell division, cell adhesion, and stressresponse (Yoshida, 2007). C-Abl shuttles between the nuclear andcytoplasmic compartments. Nuclear c-Abl is involved in cell growthinhibition and promotion of apoptosis. In contrast, the role ofcytoplasmic c-Abl is less well described. There are hints for a role inmorphogenesis and F-actin dynamics, and a role in signalling induced byextracellular stimuli like growth factors and integrin ligands.Cytoplasmic c-Abl was reported to promote mitogenesis. C-Abl mitogenicsubstrates have not yet been identified, but they are likely to includeregulators of small GTPases of the Rho family, especially Vav and Sosmembers.

The DNA-binding activity of the ubiquitously expressed ABL1 tyrosinekinase is regulated by CDC2-mediated phosphorylation, suggesting a cellcycle function for ABL1. Activity of c-Abl protein is negativelyregulated by its SH3 domain, and deletion of the SH3 domain turns ABL1into an oncogene. The c-Abl nonreceptor tyrosine kinase regulates actinresponses in nonhematopoietic cells. Some studies identify c-Abl as akey player in the signaling cascade, leading to actin reorganizationduring T-cell activation (Huang et al., 2008).

Mutations in the ABL1 gene are associated with chronic myelogenousleukemia (CML). In CML, the gene is activated by being translocatedwithin the BCR (breakpoint cluster region) gene on chromosome 22. Thisnew fusion gene, BCR-ABL, encodes an unregulated, cytoplasm targetedtyrosine kinase which allows the cells to proliferate without beingregulated by cytokines. This in turn allows the cell to become cancerous(Zhao et al., 2009). Activated c-Abl tyrosine kinase, not as a fusionprotein, plays an important role in malignant solid tumors of lung andbreast (Lin and Arlinghaus, 2008).

Recent observations indicate that c-Abl is also deregulated in solidtumors. High cytoplasmic kinase activities have been detected in breastcarcinomas and NSCLC. Overexpression is, however, not sufficient andconstitutive kinase activity required protein phosphorylation. In breastcancer cells, c-Abl phosphorylation is induced by plasma membranetyrosine kinases, including SFK, EGFR family members and the IGF-1receptor. ABL fusion proteins have not been detected in solid tumors.

ABL1 and gastric cancer—In a immunohistochemical study of ABL1expression, a wide range of normal fetal and adult human tissues and avariety of tumour types were examined. Most tumours showed focal or weakABL immunoreactivity. The most intense staining was seen inchondrosarcoma, liposarcoma, and diffuse gastric (signet ring)adenocarcinoma. In the two latter cases, ABL was also expressed on tumormicrovessels, indicating a possible role in angiogenesis.

Recent studies have revealed that infection with cagA-positiveHelicobacter pylori plays an essential role in the development ofgastric carcinoma. H. pylori blocks EGFR endocytosis and receptordegradation upon prolonged infection of gastric epithelial cells.Moreover, this inhibition occurs via a CagA-dependent, but CagAphosphorylation-independent activation of the non-receptor kinase c-Abl,which in turn phosphorylates the EGFR target site pY1173 (Bauer et al.,2009). Selective inhibition of c-Abl kinase activity by STI571 or shRNAabrogates sustained cytotoxin-associated gene A (CagA) phosphorylationand epithelial cell migration, indicating a pivotal role of c-Abl in H.pylori infection and pathogenicity (Poppe et al., 2007).

An example of kinase blockers is Imatinib (Imatinib mesylate, GLEEVEC®,STI571), the inhibitor of Bcr/Abl oncoprotein, which has become afirst-line therapy for chronic myelogenous leukemia (Pytel et al.,2009). Imatinib has been approved for the treatment of patients withadvanced gastrointestinal stromal tumour (GIST), in which KIT, atyrosine kinase receptor, is abnormally expressed (Croom and Perry,2003). Another kinase inhibitor used recently in cancer therapy isDasatinib (BMS-354825) which is specific for ABL non-receptorcytoplasmic (Pytel et al., 2009). Nilotinib is an oral second-generationbcr-abl TKI indicated for the treatment of imatinib resistant or-intolerant Ph+CML-CP and -AP in adults (Deremer et al., 2008).

SEQ ID NO 13, SEQ ID NO 14 and SEQ ID NO 15 are disclosed in WO2007/028574, CDC42 (cell division cycle 42) is a protein involved inregulation of the cell cycle. The protein is a small GTPase of theRho-subfamily, which regulates signaling pathways that control diversecellular functions including cell morphology, migration, endocytosis andcell cycle progression. CDC42 was found to be highly over-expressed inglioblastoma.

WO 2004/067023 describes MHC Class I-restricted peptides derived fromthe tumor associated antigen survivin, which peptides are capable ofbinding to Class I HLA molecules at a high affinity.

Secreted phosphoprotein 1 (SPP1), also known as bone sialoprotein I(BSP-1), early T-lymphocyte activation (ETA-1), and most commonly asosteopontin (OPN), is a human gene product, which is also conserved inother species. Osteopontin has been implicated as an important factor inbone remodeling. Specifically, research suggests that it plays a role inanchoring osteoclasts to the mineral matrix of bones. The organic partof bone is about 20% of the dry weight, and counts in, other thanosteopontin, collagen type I, osteocalcin, osteonectin, bone sialoprotein and alkaline phosphatase. Collagen type I counts for 90% of theprotein mass.

OPN binds to several integrin receptors including α4β1, α9β1, and α9β4expressed by leukocytes. These receptors have been well-established tofunction in cell adhesion, migration, and survival in these cells.Therefore, recent research efforts have focused on the role of OPN inmediating such responses.

Osteopontin is expressed in a range of immune cells, includingmacrophages, neutrophils, dendritic cells, and T and B cells, withvarying kinetics. OPN is reported in act as an immune modulator in avariety of manners. Firstly, it has chemotactic properties, whichpromote cell recruitment to inflammatory sites. It also functions as anadhesion protein, involved in cell attachment and wound healing. Inaddition, OPN mediates cell activation and cytokine production, as wellas promoting cell survival by regulating apoptosis.

Activated T cells are promoted by IL-12 to differentiate towards the Th1type, producing cytokines including IL-12 and IFNγ. OPN inhibitsproduction of the Th2 cytokine IL-10, which leads to enhanced Th1response. OPN influences cell-mediated immunity and has Th1 cytokinefunctions. It enhances B cell immunoglobulin production andproliferation. Recent studies in 2008 suggest that OPN also induces mastcell degranulation. [Nagasaka A, Matsue H, Matsushima H, et al.(February 2008). “Osteopontin is produced by mast cells and affectsIgE-mediated degranulation and migration of mast cells”. Eur. J.Immunol. 38 (2): 489-99] The researchers observed that IgE-mediatedanaphylaxis was significantly reduced in OPN knock-out mice compared towild type mice. The role of OPN in activation of macrophages has alsobeen implicated in a cancer study, when researchers discovered thatOPN-producing tumors were able to induce macrophage activation comparedto OPN-deficient tumors.

OPN is an important anti-apoptotic factor in many circumstances. OPNblocks the activation-induced cell death of macrophages and T cells aswell as fibroblasts and endothelial cells exposed to harmful stimuli.OPN prevents non-programmed cell death in inflammatory colitis.

The fact that OPN interacts with multiple cell surface receptors whichare ubiquitously expressed makes it an active player in manyphysiological and pathological processes including wound healing, boneturnover, tumorigenesis, inflammation, ischemia and immune responses.Therefore, manipulation of plasma OPN levels may be useful in thetreatment of autoimmune diseases, cancer metastasis, osteoporosis andsome forms of stress.

It has been shown that OPN drives IL-17 production; OPN is overexpressedin a variety of cancers, including lung cancer, breast cancer,colorectal cancer, stomach cancer, ovarian cancer, melanoma andmesothelioma; OPN contributes both glomerulonephritis andtubulointerstitial nephritis; and OPN is found in atheromatous plaqueswithin arteries. Thus, manipulation of plasma OPN levels may be usefulin the treatment of autoimmune diseases, cancer metastasis, osteoporosisand some forms of stress.

Human Epidermal Growth Factor Receptor 3 (ERBB3)

ERBB3 encodes a member of the epidermal growth factor receptor (EGFR)family of receptor tyrosine kinases. It is activated by neuregulins, byother ERBB and nonERBB receptors as well as by other kinases, and bynovel mechanisms. Downstream it interacts prominently with thephosphoinositol 3-kinase/AKT survival/mitogenic pathway, but also withGRB, SHC, SRC, ABL, rasGAP, SYK and the transcription regulator EBP1(Sithanandam and Anderson 413-48).

Studies of ERBB3 expression in primary cancers and of its mechanisticcontributions in cultured cells have implicated it, with varying degreesof certainty, with causation or sustenance of cancers of the breast,ovary, prostate, certain brain cells, retina, melanocytes, colon,pancreas, stomach, oral cavity and lung (Sithanandam and Anderson413-48). ERBB3 protein was detected by immunohisto-chemistry inepithelial cells throughout the gastrointestinal tract, includingsquamous epithelium of the oropharynx and esophagus, parietal cells ofthe stomach and surface enterocytes of small and large bowel. ERBB3showed increased expression in gastric cancers (Poller et al. 275-80;Sanidas et al. 935-40). Gastric cancer cell lines all expressed ERBB3and a truncated, secreted product. Strong evidence for a key role forERBB3 in gastric malignancy came from a study of poorly differentiatedsignet-ring cell gastric carcinomas (Kobayashi et al. 1294-301). Zhanget al investigated the expression of ERBB3 in gastric cancer of twopathologic types (intestinal type and diffuse type) usingimmunohistochemistry (IHC). The diffuse type of GC exhibited asignificantly higher rate of ERBB3 overexpression than the intestinaltype (26.2% vs. 5.0%, p<0.01). The selective overexpression of ERBB3 inthe two histologic types of gastric cancer is strongly associated with apoor prognosis (Zhang et al. 2112-18). ERBB3 expression wassignificantly associated with parameters involved with tumorprogression, including the depth of tumor invasion, involved lymphnodes, distant metastasis, tumor stage, and recurrent disease (Hayashiet al. 7843-49). The expression and coexpression of EGFR, c-erbB-2 andc-erbB-3 in 21 gastric cancers and 20 chronic gastritis' were examinedusing immunohistochemistry on fresh frozen tissues consideringclinicopathological variables. Generally, gastric cancer patients showeda higher incidence of EGFR, c-erbB-2 and d-erbB-3 overexpression thanthe group with chronic gastritis (81% and 43%; 38% and 45%; 35% and 20%,respectively), however, statistically significant differences were foundonly for EGFR expression (p=0.01) (Slesak et al. 2727-32).

Several approaches for therapeutic targeting of ERBB3 have been triedexperimentally. RNA aptamers to the extracellular domain of ERBB3inhibited NRG-induced ERBB3/ERBB2 heterodimerization, ERBB2phosphorylation and growth of MCF7 breast cancer cells (Chen et al.9226-31). A synthetic designer zinc finger transcription factorinhibitory to ERBB3 gene expression in A431 squamous cell carcinomacells resulted in reduced proliferation and migration, and therepression of ERBB3 expression had a bigger effect than changing ERBB2(Lund et al. 9082-91). The vitamin E isomer γ-tocotrienol inhibitedmammary cell proliferation by specific block of ERBB3 activation and ofdownstream stimulation of the PI3K/AKT pathway (Samant and Sylvester563-74). Micro-RNA 125a reduced ERBB3 RNA and protein, activation of AKTand cell growth and invasiveness of SKBR3 mammary carcinoma cells (Scottet al. 1479-86). Downregulation of ERBB3 by siRNA in breast cancer cellsabrogated their secondary resistance to tyrosine kinase inhibitors andallowed induction of apoptosis (Sergina et al. 437-41). Small inhibitoryRNA (siRNA) to ERBB3 or AKT is showing promise as a therapeutic approachto treatment of lung adenocarcinoma (Sithanandam et al. 1847-59).

Survivin (BIRC5)

Expression of BIRC5 (survivin), a member of the inhibitor of apoptosisprotein (IAP) family, is elevated in fetal tissues and in various humancancers. WO 2004/067023 describes MHC Class I-restricted peptidesderived from the tumor associated antigen survivin, which peptides arecapable of binding to Class I HLA molecules at a high affinity. Survivinseems to be capable of regulating both cellular proliferation andapoptotic cell death. Especially in glioblastoma, very high levels ofsurvivin expression are detectable (Angileri et al., 2008). It issuggested that survivin overexpression in brain gliomas might play animportant role in malignant proliferation, anti-apoptosis andangiogenesis (Zhen et al., 2005; Liu et al., 2006). Especially forglioblastoma, but also for other tumor entities, survivin expression wassignificantly associated with malignancy grade (with highest survivinexpression in glioblastoma) and shorter overall survival times comparedwith patients who had survivin-negative tumors (Kajiwara et al., 2003;Saito et al., 2007; Uematsu et al., 2005; Mellai et al., 2008; Grunda etal., 2006; Xie et al., 2006; Sasaki et al., 2002; Chakravarti et al.,2002).

Hepatitis B Core Antigen

For the Hepatitis B virus (HBV) core protein HBc immunogenic peptidesare well known (Bertoletti et al., 1993; Livingston et al., 1997). Aten-amino acid peptide from HBc may be included as a positive controlfor patients' immunocompetence and successful immunizations into cancervaccines based on the present invention.

TABLE 6 Cancer associated functions of the source proteins Cancer-associated functions of source proteins/ HLA class I TUMAP TUMAPsactivity ASPM-002 CDC2-001 MET-006 MST1R-001 PROM1-001 UCHL5-001MMP11-001 Oncofetal − − − − − − − protein (OF)/ Cancer-Testis- Antigen(CT) Tumorigenesis + + + + + ? + (cell cycle progression, proliferation)Tumor + − + + + ? + invasion, migration, metastasis Cancer- associatedsignaling Downstream Ras/MAPK, TGF-beta pathways of EGFR Cell cycle(G2/M) PI3K, PLC Similar to MET Akt (enhanced) IGF1R pathwayAnti-apoptotic (+) (+) + + − − + effects Angiogenesis − − + (+) (+) − −Cancer − − − − + − − stem-like cells Overexpression ? + + + (+) ? + inGC Overexpression + + + + + (+) + in other cancers Poorprognosis + + + + + ? + Advanced − ? + ? − − − stages Cancer- associatedfunctions of HLA source proteins/ HLA class I class II TUMAP TUMAPsTUMAP activity SMC4-001 PPAP2C-001 AVL9-001 UQCRB-001 ABL1-001 NUF2-001BIRC5-002 Oncofetal − − ? − − CT OF protein (OF)/ Cancer-Testis- Antigen(CT) Tumorigenesis − + ? − + + + (cell cycle progression, proliferation)Tumor − − ? − + − − invasion, migration, metastasis Cancer- associatedEGFR, PI3K, signaling STAT3, +1, Rac/JNK, Inhibition of pathways mitosisCell cycle ? − Erk5, eta-catenin cell division apoptosis Anti-apoptotic− − ? − + − + effects Angiogenesis − − ? − + − − Cancer (+) (+) ? − −− + stem-like cells Overexpression ? ? ? ? + + + in GCOverexpression + + ? + + + + in other cancers Poor prognosis − ? ?− + + + Advanced − + ? − − − + stages Ranking “−” < “(+)” < “+”; “?”means that the situation is currently unknown

As it can be seen from Table 6, the person skilled in the art can easilyadapt the composition of the application at hand to the patient and/ortumor specific and select the TUMAPs accordingly.

In a preferred embodiment of the invention the pharmaceuticalcomposition comprises at least two peptides, one of them containing anamino acid sequence according to SEQ ID NO: 1 and containing further apeptide containing an amino acid sequence according to SEQ ID NO: 11.

In a preferred embodiment of the invention the pharmaceuticalcomposition comprises at least two peptides, one containing an aminoacid sequence according to SEQ ID NO: 1 and an amino acid sequenceaccording to SEQ ID NO: 2 and/or SEQ ID NO: 11.

In a preferred embodiment of the invention the pharmaceuticalcomposition comprises at least two peptides containing an amino acidsequence according to SEQ ID NO: 3 and an amino acid sequence accordingto SEQ ID NO: 2 and/or SEQ ID NO: 11.

In a preferred embodiment of the invention the pharmaceuticalcomposition comprises at least two peptides containing an amino acidsequence according to SEQ ID NO: 1 and an amino acid sequence accordingto SEQ ID NO: 7 and optionally SEQ ID NO: 11.

In a preferred embodiment of the invention the pharmaceuticalcomposition comprises at least two peptides containing an amino acidsequence according to SEQ ID NO: 2 and an amino acid sequence accordingto SEQ ID NO: 7 and optionally SEQ ID NO: 11.

In a preferred embodiment of the invention the pharmaceuticalcomposition comprises at least two peptides containing an amino acidsequence according to SEQ ID NO: 3 and an amino acid sequence accordingto SEQ ID NO: 7 and optionally SEQ ID NO: 11.

In an even more preferred embodiment the pharmaceutical compositioncomprises at least one more peptides containing an amino acid sequenceselected from the group consisting of SEQ ID NO 2 to SEQ ID NO 10 andSEQ ID NO: 11 to SEQ ID NO: 22 and SEQ ID NO 24 and/or an amino acidsequence that is at least 85% identical to that of SEQ ID NO 2 to SEQ IDNO 10 and SEQ ID NO: 11 to SEQ ID NO: 22 and SEQ ID NO 24 and/or apolynucleotide containing a nucleic acid encoding SEQ ID NO 2 to SEQ IDNO 10 and SEQ ID NO: 11 to SEQ ID NO: 22 SEQ ID NO 24 or the variantamino acid sequence, and a pharmaceutically acceptable carrier.

Further preferred embodiments of the invention comprise at least 3, 4,5, 6, 7, 8, 9, 10, 11 12, 13, 14, 15, 16, 17 or 18 peptides containingan amino acid sequence selected from the group consisting of SEQ ID NO 1to SEQ ID NO 10 and SEQ ID NO: 11 to SEQ ID NO: 22 SEQ ID NO 24 and/oran amino acid sequence that is at least 85% identical to that of SEQ IDNO 1 to SEQ ID NO 10 and/or a polynucleotide containing a nucleic acidencoding SEQ ID NO 1 to SEQ ID NO 10 and SEQ ID NO: 11 to SEQ ID NO: 22SEQ ID NO 24 or the variant amino acid sequence, and a pharmaceuticallyacceptable carrier.

The pharmaceutical composition can furthermore contain additionalpeptides and/or excipients to be more effective, as will be furtherexplained below.

By a “variant amino acid sequence” of the given amino acid sequence theinventors mean that the side chains of, for example, one or two of theamino acid residues are altered (for example by replacing them with theside chain of another naturally occurring amino acid residue or someother side chain) such that the peptide is still able to bind to an HLAmolecule in substantially the same way as a peptide consisting of thegiven amino acid sequence. For example, a peptide may be modified sothat it at least maintains, if not improves, the ability to interactwith and bind a suitable MHC molecule, such as HLA-A or -DR, and so thatit at least maintains, if not improves, the ability to generateactivated CTL which can recognise and kill cells which express apolypeptide containing an amino acid sequence as defined in the aspectsof the invention. As can be derived from the database, certain positionsof HLA-A binding peptides are typically anchor residues forming a coresequence fitting to the binding motif of the HLA binding groove.

Those amino acid residues that are not essential to interact with theT-cell receptor can be modified by replacement with another amino acidwhose incorporation does not substantially affect T-cell reactivity anddoes not eliminate binding to the relevant MHC. Thus, apart from theproviso given, the peptide of the invention may be any peptide (by whichterm the inventors include oligopeptide or polypeptide) which includesthe amino acid sequences or a portion or variant thereof as given.

It is furthermore known for MHC-class II presented peptides that thesepeptides are composed of a “core sequence” having a certain HLA-specificamino acid motif and, optionally, N- and/or C-terminal extensions thatdo not interfere with the function of the core sequence (i.e. are deemedas irrelevant for the interaction of the peptide and the T-cell). The N-and/or C-terminal extensions can, for example, be from 1 to 10 aminoacids in length, respectively. These peptides can be used eitherdirectly to load MHC class II molecules or the sequence can be clonedinto the vectors according to the description herein below. As thesepeptides form the final product of the processing of larger peptideswithin the cell, longer peptides can be used as well. The peptides ofthe invention may be of any size, but typically they may be less than100,000 in molecular weight, preferably less than 50,000, morepreferably less than 10,000, more preferably less than 5,000, morepreferably less than 2,500 and typically about 1000 to 2000. In terms ofthe number of amino acid residues, the peptides of the invention mayhave fewer than 1000 residues, preferably fewer than 500 residues, morepreferably fewer than 100 residues. Accordingly the present inventionalso provides compositions of peptides and variants thereof wherein thepeptide or variant has an overall length of from 8 to 100, preferablyfrom 8 to 30, and most preferred from 8 to 17, namely 8, 9, 10, 11, 12,13, 14, 15 or 16 amino acids.

Preferred are peptides have a core sequence selected from a groupconsisting of SEQ ID NO 11 to SEQ ID NO: 22 and SEQ ID NO 24 withextensions of 1 to 10 amino acids on the C-terminal and/or theN-terminal, more preferred the overall number of these flanking aminoacids is 1 to 12, more preferred 1 to 10, more preferred 1 to 8, morepreferred 1 to 6, wherein the flanking amino acids can be distributed inany ratio to the C-terminus and the N-terminus (for example all flankingamino acids can be added to one terminus, or the amino acids can beadded equally to both termini or in any other ratio), provided that thepeptide is still able to bind to an HLA molecule in the same way as saidpeptide according to any of the SEQ ID NO 11 to SEQ ID NO: 22 and SEQ IDNO 24.

Correspondingly, variants that induce T-cells cross-reacting with apeptide of the invention are often length variants.

If a peptide is longer than around 12 amino acid residues it is useddirectly to bind to a MHC class II molecule. It is preferred that theresidues that flank the core HLA binding region do not substantiallyaffect the ability of the peptide to bind specifically to the bindinggroove of the MHC class II molecule or to present the peptide to theCTL. However, as already indicated above, it will be appreciated thatlarger peptides may be used, especially when encoded by apolynucleotide, since these larger peptides may be fragmented bysuitable antigen-presenting cells. Furthermore the flanking amino acidscan reduce the speed of peptide degradation in vivo so that the amountof the actual peptide available to the CTLs is higher compared to thepeptide without flanking amino acids.

It is also possible, that MHC class I epitopes, although usually from 8to 10 amino acids long, are generated by peptide processing from longerpeptides or proteins that include the actual epitope. Similar to MHCclass II epitopes, it is preferred that the flanking residues ofelongated precursor peptides upstream and/or downstream of the N- andC-terminus, of the actual epitope do not substantially affect thepresentation of the peptide to the CTL nor mask the sites forproteolytic cleavage necessary to yield the actual epitope mediated byprocessing of the elongated peptide.

Preferred are peptides with a core sequence consisting of SEQ ID NO 1 toSEQ ID NO 10 and SEQ ID 11 with extensions of 1 to 10 amino acids on theC-terminal and/or the N-terminal, more preferred the overall number ofthese flanking amino acids is 1 to 12, more preferred 1 to 10, morepreferred 1 to 8, more preferred 1 to 6, wherein the flanking aminoacids can be distributed in any ratio to the C-terminus and theN-terminus (for example all flanking amino acids can be added to oneterminus, or the amino acids can be added equally to both termini or inany other ratio), provided that the peptide is still able to bind to anHLA molecule in the same way as said peptide according to any of the ofSEQ ID NO 1 to SEQ ID NO 10 and SEQ ID NO: 11.

Accordingly the present invention also provides peptides and variants ofMHC class I epitopes having an overall length of not more than 100,preferably not more than 30, and most preferred from 8 to 18 namely 8,9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human MHC class I orII. Binding of a peptide or a variant to a MHC complex may be tested bymethods known in the art, for example those described in the examples ofthe present invention below or those described in the literature fordifferent MHC class II alleles (e.g. Vogt A B, Kropshofer H, KalbacherH, Kalbus M, Rammensee H G, Coligan J E, Martin R; Ligand motifs ofHLA-DRB5*0101 and DRB1*1501 molecules delineated from self-peptides; J.Immunol. 1994; 153(4):1665-1673; Malcherek G, Gnau V, Stevanovic S,Rammensee H G, Jung G, Melms A; Analysis of allele-specific contactsites of natural HLA-DR17 ligands; J. Immunol. 1994; 153(3):1141-1149;Manici S, Sturniolo T, Imro M A, Hammer J, Sinigaglia F, Noppen C,Spagnoli G, Mazzi B, Bellone M, Dellabona P, Protti M P; Melanoma cellspresent a MAGE-3 epitope to CD4(+) cytotoxic T cells in association withhistocompatibility leukocyte antigen DR11; J Exp Med. 1999; 189(5):871-876; Hammer J, Gallazzi F, Bono E, Karr R W, Guenot J, Valsasnini P,Nagy Z A, Sinigaglia F; Peptide binding specificity of HLA-DR4molecules: correlation with rheumatoid arthritis association; J Exp Med.1995 181(5):1847-1855; Tompkins S M, Rota P A, Moore J C, Jensen P E; Aeuropium fluoroimmunoassay for measuring binding of antigen to class IIMHC glycoproteins; J Immunol Methods. 1993; 163(2): 209-216; Boyton R J,Lohmann T, Londei M, Kalbacher H, Halder T, Frater A J, Douek D C,Leslie D G, Flavell R A, Altmann D M; Glutamic acid decarboxylase Tlymphocyte responses associated with susceptibility or resistance totype I diabetes: analysis in disease discordant human twins, non-obesediabetic mice and HLA-DQ transgenic mice; Int Immunol. 1998(12):1765-1776).

Peptides of the present invention may have additional N- and/orC-terminally located stretches of amino acids that do not necessarilyform part of the peptide that functions as the actual epitope for MHCmolecules but may, nevertheless, be important to provide for anefficient introduction of the peptide according to the present inventioninto the cells (see above). In one embodiment of the present invention,the peptide of the present invention is a fusion protein whichcomprises, for example, the 80 N-terminal amino acids of the HLA-DRantigen-associated invariant chain (p33, in the following “Ii”) asderived from the NCBI, GenBank Accession-number X00497 (Strubin, M.,Mach, B. and Long, E. O. The complete sequence of the mRNA for theHLA-DR-associated invariant chain reveals a polypeptide with an unusualtransmembrane polarity EMBO J. 3 (4), 869-872 (1984)).

The present invention also provides a pharmaceutical compositioncomprising at least one of the peptides of the present invention,wherein the peptides have an overall length of not more than 100,preferably not more than 30, and most preferred from 8 to 17 or 9, 10,11, 12, 13, 14, 15, or 16 amino acids.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules to elicit a stronger immuneresponse. Methods for such an optimisation of a peptide sequence arewell known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

Thus, according to another aspect the invention provides apharmaceutical composition, wherein at least one peptide or variantincludes non-peptide bonds.

In a reverse peptide bond amino acid residues are not joined by peptide(—CO—NH—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) J. Immunol. 159,3230-3237, incorporated herein by reference. This approach involvesmaking pseudopeptides containing changes involving the backbone, and notthe orientation of side chains. Meziere et al (1997) show that for MHCand T helper cell responses, these pseudopeptides are useful.Retro-inverse peptides, containing NH—CO bonds instead of CO—NH peptidebonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains that involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences of the invention described above maybe synthesized with additional chemical groups present at their aminoand/or carboxy termini, to enhance, for example, the stability,bioavailability, and/or affinity of the peptides. For example,hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonylgroups may be added to the peptides' amino termini. Likewise, an acetylgroup or a 9-fluorenylmethoxy-carbonyl group may be placed at thepeptides' amino termini. Additionally, e.g. the hydrophobic group,t-butyloxycarbonyl, or an amido group may be added to the peptides'carboxy termini.

Further, all peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarised e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2005, which is incorporatedherein by reference. Chemical modification of amino acids includes butis not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley & Sons NY 1995-2000) for more extensivemethodology relating to chemical modification of proteins.

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethyleneglycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized e.g. using theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Luet al (1981) J. Org. Chem. 46, 3433 and references therein.

Purification may be effected by any one, or a combination of, techniquessuch as recristallisation, size exclusion chromatography, ion-exchangechromatography, hydrophobic interaction chromatography and (usually)reverse-phase high performance liquid chromatography using e.g.acetonitril/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

A further aspect of the invention provides a nucleic acid (e.g.polynucleotide) encoding a peptide or variant of the invention. Thepolynucleotide may be e.g. DNA, cDNA, PNA, CNA, RNA, either single-and/or double-stranded, or native or stabilised forms ofpolynucleotides, such as e.g. polynucleotides with a phosphorothiatebackbone, or combinations thereof and it may or may not contain intronsso long as it codes for the peptide. Of course, it is only peptidescontaining naturally occurring amino acid residues joined by naturallyoccurring peptide bonds are encodable by a polynucleotide. A stillfurther aspect of the invention provides an expression vector capable ofexpressing a polypeptide according to the invention. Expression vectorsfor different cell types are well known in the art and can be selectedwithout undue experimentation.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognised bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Guidance can be found e.g. in Sambrook et al (1989)Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

In a particularly preferred embodiment of the invention, however, thepharmaceutical composition comprises at least two peptides consisting ofamino acid sequences according to SEQ ID NO 1 to SEQ ID NO 12.

The optimum amount of each peptide to be included in the vaccine and theoptimum dosing regimen can be determined by one skilled in the artwithout undue experimentation. For example, the peptide or its variantmay be prepared for intravenous (i.v.) injection, sub-cutaneous (s.c.)injection, intradermal (i.d.) injection, intraperitoneal (i.p.)injection, intramuscular (i.m.) injection. Preferred routes of peptideinjection are s.c., i.d., i.p., i.m., and i.v. Preferred routes of DNAinjection are i.d., i.m., s.c., i.p. and i.v. Doses of e.g. between 1and 500 mg and 50 μg and 1.5 mg, preferably 125 μg to 500 μg, of peptideor DNA may be given and will depend from the respective peptide or DNA.Doses of this range were successfully used in previous trials (BrunsvigP F, Aamdal S, Gjertsen M K, Kvalheim G, Markowski-Grimsrud C J, Sve I,Dyrhaug M, Trachsel S, Møller M, Eriksen J A, Gaudernack G; Telomerasepeptide vaccination: a phase I/II study in patients with non-small celllung cancer; Cancer Immunol Immunother. 2006; 55(12):1553-1564; M.Staehler, A. Stenzl, P. Y. Dietrich, T. Eisen, A. Haferkamp, J. Beck, A.Mayer, S. Walter, H. Singh, J. Frisch, C. G. Stief; An open label studyto evaluate the safety and immunogenicity of the peptide based cancervaccine IMA901, ASCO meeting 2007; Abstract No 3017).

Pharmaceutical compositions of the present invention may be compiledsuch that the selection, number and/or amount of peptides present in thecomposition is/are tissue, cancer, and/or patient-specific. For instancethe exact selection of peptides can be guided by expression patterns ofthe parent proteins in a given tissue to avoid side effects. Theselection may be dependent from the specific type of cancer that thepatient to be treated is suffering from as well as the status of thedisease, earlier treatment regimens, the immune status of the patient,and, of course, the HLA-haplotype of the patient. Furthermore, thevaccine according to the invention can contain individualisedcomponents, according to personal needs of the particular patient.Examples are different amounts of peptides according to the expressionof the related TAAs in the particular patient, unwanted side-effects dueto personal allergies or other treatments, and adjustments for secondarytreatments following a first round or scheme of treatment.

For compositions to be used as a vaccine for GBM for example, peptideswhose parent proteins are expressed in high amounts in normal tissueswill be avoided or be present in low amounts in the composition of theinvention. On the other hand, if it is known that the tumor of a patientexpresses high amounts of a certain protein the respectivepharmaceutical composition for treatment of this cancer may be presentin high amounts and/or more than one peptide specific for thisparticular protein or pathway of this protein may be included. Theperson of skill will be able to select preferred combinations ofimmunogenic peptides by testing, for example, the generation of T-cellsin vitro as well as their efficiency and overall presence, theproliferation, affinity and expansion of certain T-cells for certainpeptides, and the functionality of the T-cells, e.g. by analysing theIFN-gamma production (see also examples below). Usually, the mostefficient peptides are then combined as a vaccine for the purposes asdescribed above.

A suitable vaccine will preferably contain from 1 to 20 peptides, morepreferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 different peptides, further preferred 6, 7, 8, 9, 10 11, 12,13, or 14 different peptides, and most preferably 10, 11, 12, 13 or 14different peptides. The length of the peptide for use in a cancervaccine may be any suitable length, as disclosed herein. In particular,it may be a suitable 9-mer peptide or a suitable 8-mer or 9-mer or10-mer or 11-mer peptide or 12-mer, 13-mer, 14-mer or 15-mer. Longerpeptides may also be suitable, 9-mer or 10-mer peptides as described inthe attached Tables 4 and 5 are preferred for MHC class I-peptides,while 12- to 15-mers are preferred for MHC class II peptides.

The peptide(s) constitute(s) a tumor or cancer vaccine. It may beadministered directly into the patient, into the affected organ orsystemically, or applied ex vivo to cells derived from the patient or ahuman cell line which are subsequently administered to the patient, orused in vitro to select a subpopulation from immune cells derived fromthe patient, which are then re-administered to the patient.

The peptide may be substantially pure, or combined with animmune-stimulating adjuvant (see below) or used in combination withimmune-stimulatory cytokines, or may be administered with a suitabledelivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and Longenecker et al (1993) Ann. NYAcad. Sci. 690, 276-291). The peptide may also be tagged, or be a fusionprotein, or be a hybrid molecule. The peptides whose sequence is givenin the present invention are expected to stimulate CD4 T cells or CD8CTL. However, stimulation is more efficient in the presence of helpprovided by T-cells positive for the opposite CD. Thus, for MHC Class IIepitopes that stimulate CD4 T cells the fusion partner or sections of ahybrid molecule suitably provide epitopes which stimulate CD8-positiveT-cells. On the other hand, for MHC Class I epitopes which stimulate CD8CTL the fusion partner or sections of a hybrid molecule suitably provideepitopes which stimulate CD4-positive T cells. CD4- and CD8-stimulatingepitopes are well known in the art and include those identified in thepresent invention.

Pharmaceutically acceptable carriers are well known and are usuallyliquids, in which an active therapeutic agent is formulated. The carriergenerally does not provide any pharmacological activity to theformulation, though it may provide chemical and/or biological stability,release characteristics, and the like. Exemplary formulations can befound, for example, in Alfonso R. Gennaro. Remington: The Science andPractice of Pharmacy, 20th Edition. Baltimore, Md.: Lippincott Williams& Wilkins, 2000 and include, but are not limited to, saline, water,buffered water, 0.3% glycine, hyaluronic acid, dextrose and the like.Recently, it was found that certain fat emulsions, which have been inuse for many years for intravenous nutrition of human patients, can alsoact as a vehicle for peptides. Two examples of such emulsions are theavailable commercial fat emulsions known as Intralipid and Lipofundin.“Intralipid” is a registered trademark of Kabi Pharmacia, Sweden, for afat emulsion for intravenous nutrition, described in U.S. Pat. No.3,169,094. “Lipofundin” is a registered trademark of B. Braun Melsungen,Germany. Both contain soybean oil as fat (100 or 200 g in 1,000 mldistilled water: 10% or 20%, respectively). Egg-yolk phospholipids areused as emulsifiers in Intralipid (12 g/l distilled water) and egg-yolklecithin in Lipofundin (12 g/l distilled water). Isotonicity resultsfrom the addition of glycerol (25 g/l) both in Intralipid andLipofundin.

To elicit an immune response it is usually necessary to includeadjuvants that render the composition more immunogenic. Thus in apreferred embodiment of the invention the pharmaceutical compositionfurther comprises at least one suitable adjuvant.

Suitable adjuvants include, but are not limited to, 1018 ISS, aluminiumsalts, Amplivax®, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellinor TLR5 ligands derived from flagellin, FLT3 ligand, GM-CSF, IC30, IC31,Imiquimod (ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2,IL-13, IL-21, Interferon-alpha or -beta, or pegylated derivativesthereof, IS Patch, ISS, ISCOMATRIX®, ISCOMs, Juvlmmune, LipoVac, MALP2,MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206,Montanide ISA 50V, Montanide ISA-51, water-in-oil and oil-in-wateremulsions, OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vectorsystem, poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Aucouturieret al., 2001; Allison and Krummel, 1995). Also cytokines may be used.Several cytokines have been directly linked to influencing dendriticcell migration to lymphoid tissues (e.g., TNF-), accelerating thematuration of dendritic cells into efficient antigen-presenting cellsfor T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No.5,849,589, specifically incorporated herein by reference in itsentirety) and acting as immunoadjuvants (e.g., IL-12, IL-15, IL-23,IL-7, IFN-alpha. IFN-beta) (Gabrilovich et al., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of T_(H1) cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The T_(H1) biasinduced by TLR9 stimulation is maintained even in the presence ofvaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA)that normally promote a T_(H2) bias. CpG oligonucleotides show evengreater adjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such aspoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC-R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, Bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil,vardenafil, sorafenib, temozolomide, temsirolimus, XL-999, CP-547632,pazopanib, VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodiestargeting key structures of the immune system (e.g. anti-CD40,anti-TGFbeta, anti-TNFalpha receptor) and SC58175, which may acttherapeutically and/or as an adjuvant. The amounts and concentrations ofadjuvants and additives useful in the context of the present inventioncan readily be determined by the skilled artisan without undueexperimentation.

Preferred adjuvants are imiquimod, resiquimod, GM-CSF, cyclophosphamide,sunitinib, bevacizumab, interferon-alpha, CpG oligonucleotides andderivates, poly-(I:C) and derivates, RNA, sildenafil, and particulateformulations with PLG or virosomes.

In a preferred embodiment the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), imiquimod, resiquimod, andinterferon-alpha.

In a preferred embodiment of the pharmaceutical composition according tothe invention, the adjuvant is imiquimod or resiquimod.

In a preferred embodiment of the pharmaceutical composition according tothe invention, the adjuvant is imiquimod or resimiquimod.

In a preferred embodiment of the pharmaceutical composition according tothe invention, the adjuvant is the combination of GM-CSF and imiquimod.

Compositions of the present invention may be used for parenteraladministration, such as subcutaneous, intradermal, intramuscular,intraperitoneal or for oral administration. For this, the peptides andoptionally other molecules are dissolved or suspended in apharmaceutically acceptable, preferably aqueous carrier. In addition,the composition can contain excipients, such as buffers, binding agents,blasting agents, diluents, flavours, lubricants, etc. The peptides canalso be administered together with immune stimulating substances, suchas cytokines. An extensive listing of excipients that can be used insuch a composition, can be, for example, taken from A. Kibbe, Handbookof Pharmaceutical Excipients, 3^(rd) Ed. 2000, American PharmaceuticalAssociation and pharmaceutical press. The composition can be used for aprevention, prophylaxis and/or therapy of adenomateous or cancerousdiseases, preferably CRC.

Preferred formulations can be found, for example, in EP2113253.

Cytotoxic T-cells (CTLs) recognise an antigen in the form of a peptidebound to an MHC molecule rather than the intact foreign antigen itself.The MHC molecule itself is located at the cell surface of an antigenpresenting cell. Thus, an activation of CTLs is only possible if atrimeric complex of peptide antigen, MHC molecule, and APC is present.Correspondingly, it may enhance the immune response if not only thepeptide is used for activation of CTLs but if additionally APCs with therespective MHC molecule are added.

Therefore, in a preferred embodiment the pharmaceutical compositionaccording to the present invention additionally contains at least oneantigen-presenting cell.

The antigen-presenting cell (or stimulator cell) typically has an MHCclass I or II molecule on its surface and in one embodiment issubstantially incapable of loading itself the MHC class I or II moleculewith the selected antigen. As it is described in more detail below, theMHC class I or II molecule may readily be loaded with the selectedantigen in vitro.

Preferably the mammalian cell lacks or has a reduced level or hasreduced function of the TAP peptide transporter. Suitable cells whichlack the TAP peptide transporter include T2, a human peptide loadingdeficient cell line that is available from the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md. 20852, USA underCatalogue No CRL 1992; TAP-deficient cell lines such as T2 can be usedas APCs, and due to the lack of TAP nearly all peptides presented by MHCclass I will be the peptides under scrutiny used for externally loadingthe empty MHC class I molecules of these cell lines, hence all effectswill clearly attribute to the used peptides.

Preferably, the antigen presenting cells are dendritic cells. Suitably,the dendritic cells are autologous dendritic cells which are pulsed withan antigenic peptide. The antigenic peptide may be any suitableantigenic peptide which gives rise to an appropriate T-cell response.T-cell therapy using autologous dendritic cells pulsed with peptidesfrom a tumor associated antigen is disclosed in Murphy et al (1996) TheProstate 29, 371-380, and Tjua et al (1997) The Prostate 32, 272-278.

Thus, in a preferred embodiment of the present invention thepharmaceutical composition containing at least one antigen presentingcell is pulsed or loaded with the peptide, for instance by the methodshown in example 5.

As an alternative the antigen presenting cell comprises an expressionconstruct encoding the peptide. The polynucleotide may be any suitablepolynucleotide and it is preferred that it is capable of transducing thedendritic cell thus resulting in the presentation of a peptide andinduction of immunity.

Conveniently, a nucleic acid of the invention may be comprised in aviral polynucleotide or virus. For example, adenovirus-transduceddendritic cells have been shown to induce antigen-specific antitumorimmunity in relation to MUC1 (see Gong et al (1997) Gene Ther. 4,1023-1028). Similarly, adenovirus-based systems may be used (see, forexample, Wan et al (1997) Hum. Gene Ther. 8, 1355-1363); retroviralsystems may be used (Koch et al., 2006) J. Exp. Med. 186, 1213-1221 andSzabolcs et al (1997) Blood particle-mediated transfer to dendriticcells may also be used {Tuting 1997} Eur. J. Immunol. 27, 2702-2707);and RNA may also be used (Ashley et al., 2007) J. Exp. Med. 186, 11771182).

Generally, a pharmaceutical composition of the invention containing (a)nucleic acid(s) of the invention can be administered in a similar manneras those containing peptide(s) of the invention, e.g. intravenously,intra-arterially, intra-peritoneally, intramuscularly, intradermally,intratumorally, orally, dermally, nasally, buccally, rectally,vaginally, by inhalation, or by topical administration.

Due to evasion mechanisms a tumor often develops resistance to thetreatment. The drug resistance may occur during treatment and manifestsitself in metastases and recurring tumors. To avoid such a drugresistance a tumor is commonly treated by a combination of drugs andmetastases and tumors recurring after a disease free period of timeoften require a different combination. Therefore, in one aspect of theinvention the pharmaceutical composition is administered in conjunctionwith a second anticancer agent. The second agent may be administeredbefore after or simultaneously with the pharmaceutical composition ofthe invention. A simultaneous administration can e.g. be achieved bymixing the pharmaceutical composition of the invention with the secondanticancer agent if chemical properties are compatible. Another way of asimultaneous administration is the administration of the composition andanticancer agent on the same day independently from the route ofadministration such that the pharmaceutical composition of the inventionmay be e.g. injected while the second anticancer agent is for instancegiven orally. The pharmaceutical composition and second anticancer agentmay also be administered within the same treatment course but ondifferent days and/or within separate treatment courses.

In another aspect the present invention provides a method for treatingor preventing a cancer in a patient comprising administering to thepatient a therapeutically effective amount any one of the pharmaceuticalcompositions of the invention.

A therapeutically effective amount will be an amount sufficient toinduce an immune response, in particular an activation of asubpopulation of CTLs. A person skilled in the art may easily determinewhether an amount is effective by using standard immunological methods,such as those provided in the examples of the present specifications.Another way of monitoring the effect of a certain amount of thepharmaceutical composition is to observe the growth of the tumor treatedand/or its recurrence.

In a particularly preferred embodiment of the present invention thepharmaceutical composition is used as an anti-cancer vaccine.

The composition containing peptides or peptide-encoding nucleic acidscan also constitute a tumor or cancer vaccine. It may be administereddirectly into the patient, into the affected organ or systemically, orapplied ex vivo to cells derived from the patient or a human cell linewhich are subsequently administered to the patient, or used in vitro toselect a subpopulation from immune cells derived from the patient, whichare then re-administered to the patient.

The composition of the invention may be used in a method for treating ofor used as a vaccine for cancer. The cancer may be prostate carcinoma,oral cavity carcinomas, oral squamous carcinoma (OSCC), acute myeloidleukemia (AML) (Qian et al., 2009), H. pylori-induced MALT lymphoma(Banerjee et al., 2000), colon carcinoma/colorectal cancer,glioblastoma, non-small-cell lung cancer (NSCLC), cervical carcinoma,human breast cancer, prostate cancer, colon cancer, pancreatic cancers,pancreatic ductal adenocarcinoma, ovarian cancer, hepatocellularcarcinoma, liver cancer, brain tumors of different phenotypes, leukemiassuch as acute lymphoblastic leukemia, ALL, lung cancer, Ewing's sarcoma,endometrial cancer, head and neck squamous cell carcinoma, epithelialcancer of the larynx, oesophageal carcinoma, oral carcinoma, carcinomaof the urinary bladder, ovarian carcinomas, renal cell carcinoma,atypical meningioma, papillary thyroid carcinoma, brain tumors, salivaryduct carcinoma, extranodal T/NK-cell lymphomas, Non-Hodgkins Lymphomaand malignant solid tumors of the lung and breast, preferably the canceris gastric cancer.

In the most preferred embodiment of the method of treatment or vaccineaccording to the invention, the vaccine is a multiple peptide tumorvaccine for treatment of GC. Preferably, the vaccine comprises a set oftumor-associated peptides selected from SEQ ID No. 1 to SEQ ID No. 11which are located and have been identified on primary GC cells. This setincludes HLA class I and class II peptides. The peptide set can alsocontain at least one peptide, such as from HBV core antigen (SEQ ID 23),used as a positive control peptide serving as immune marker to test theefficiency of the intradermal administration. In one particularembodiment, the vaccine consists of 11 individual peptides (according toSEQ ID No. 1 to SEQ ID No. 11) with between about 1500 μg to about 75μg, preferably between about 1000 μg to about 175 μg and more preferredbetween about 500 μg to about 600 μg, and most preferred about 578 μg ofeach peptide, all of which may be purified by HPLC and ion exchangechromatography and appear as a white to off-white powder. Thelyophilisate is preferably dissolved in sodium hydrogen carbonate, andis used for intradermal injection within 30 min after reconstitution atroom temperature. According to the present invention, preferred amountsof peptides can vary between about 0.1 and 100 mg, preferably betweenabout 0.1 and 1 mg, and most preferred between about 300 μg and 800 μgper 500 μl of solution. Herein, the term “about” shall mean +/−10percent of the given value, if not stated differently. The person ofskill will be able to adjust the actual amount of peptide to be usedbased on several factors, such as, for example, the immune status of theindividual patient and/or the amount of TUMAP that is presented in aparticular type of cancer. The peptides of the present invention mightbe provided in other suitable forms (sterile solutions, etc.) instead ofa lyophilisate.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt.

As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH₂ group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, basic salts of acid moietieswhich may be present on a peptide are prepared using a pharmaceuticallyacceptable base such as sodium hydroxide, potassium hydroxide, ammoniumhydroxide, calcium hydroxide, trimmethylamine or the like.

In an especially preferred embodiment the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), ammonium orhydrochloric acid (chlorides).

In another embodiment, a pharmaceutical composition of the presentinvention may include sugars, sugar alcohols, aminoacids such a glycin,arginine, glutaminic acid and others as framework former. The sugars maybe mono-, di- or trisaccharide. These sugars may be used alone, as wellas in combination with sugar alcohols. Examples of sugars includeglucose, mannose, galactose, fructose or sorbose as monosaccharides,saccharose, lactose, maltose or trehalose as disaccharides and raffinoseas a trisaccharid. A sugar alcohol may be, for example, mannitose.Preferred ingredients are saccharose, lactose, maltose, trehalose,mannit and/or sorbit, and more preferably, mannitol.

Furthermore pharmaceutical compositions of the present invention mayinclude physiological well tolerated excipients (see Handbook ofPharmaceutical Excipients, 5^(th) ed., edited by Raymond Rowe, PaulSheskey and Sian Owen, Pharmaceutical Press (2006)), such asantioxidants like ascorbic acid or glutathione, preserving agents suchas phenole, m-cresole, methyl- or propylparabene, chlorobutanol,thiomersal or benzalkoniumchloride, stabilizer, framework former such assaccharose, lactose, maltose, trehalose, mannitose, mannit and/orsorbit, mannit and/or lactose and solubilizer such aspolyethyleneglycols (PEG), i.e. PEG 3000, 3350, 4000 or 6000, orcyclodextrines, i.e. hydroxypropyle-β-cyclodextrine,sulfobutylethyl-β-cyclodextrine or γ cyclodextrine, or dextranes orpoloxaomers, i.e. poloxaomer 407, poloxamer 188, or Tween 20, Tween 80.In a preferred embodiment pharmaceutical compositions of the presentinvention include one or more well tolerated excipients, selected fromthe group consisting of antioxidants, framework formers and stabilizers.

The acceptable pH-range is pH 2-12 for intravenous and intramuscularadministration, but subcutaneously the range is reduced to 2.7-9.0 asthe rate of in vivo dilution is reduced resulting in more potential forirradiation at the injection site. Strickley Robert G., Pharm. Res., 21,NO:2, 201-230 (2004).

The pharmaceutical preparation of the present invention comprisingpeptides, and/or nucleic acid(s) according to the invention isadministered to a patient that suffers from an adenomateous or cancerousdisease that is associated with the respective peptide or antigen. Bythis, a T cell-mediated immune response can be triggered.

Preferred is a pharmaceutical composition according to the invention,wherein the amount of (in particular tumor associated) peptide(s), ofnucleic acid(s) according to the invention or expression vector(s)according to the invention as present in the composition is/are tissue,cancer, and/or patient-specific.

In another embodiment of the invention the vaccine is a nucleic acidvaccine. It is known that inoculation with a nucleic acid vaccine, suchas a DNA vaccine, encoding a polypeptide leads to a T-cell response. Itmay be administered directly into the patient, into the affected organor systemically, or applied ex vivo to cells derived from the patient ora human cell line which are subsequently administered to the patient, orused in vitro to select a subpopulation from immune cells derived fromthe patient, which are then re-administered to the patient. If thenucleic acid is administered to cells in vitro, it may be useful for thecells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2 or GM-CSF. The nucleic acid(s) may besubstantially pure, or combined with an immune-stimulating adjuvant, orused in combination with immune-stimulatory cytokines, or beadministered with a suitable delivery system, for example liposomes. Thenucleic acid vaccine may also be administered with an adjuvant such asthose described for peptide vaccines above. It is preferred if thenucleic acid vaccine is administered without adjuvant.

The polynucleotide may be substantially pure, or contained in a suitablevector or delivery system. Suitable vectors and delivery systems includeviral, such as systems based on adenovirus, vaccinia virus,retroviruses, herpes virus, adeno-associated virus or hybrids containingelements of more than one virus. Non-viral delivery systems includecationic lipids and cationic polymers as are well known in the art ofDNA delivery. Physical delivery, such as via a “gene-gun”, may also beused. The peptide or peptide encoded by the nucleic acid may be a fusionprotein, for example with an epitope from tetanus toxoid whichstimulates CD4-positive T-cells.

Suitably, any nucleic acid administered to the patient is sterile andpyrogen free. Naked DNA may be given intramuscularly or intradermally orsubcutaneously. Conveniently, the nucleic acid vaccine may comprise anysuitable nucleic acid delivery means. The nucleic acid, preferably DNA,may also be delivered in a liposome or as part of a viral vectordelivery system. It is preferred if the nucleic acid vaccine, such asDNA vaccine, is administered into the muscle, whilst peptide vaccinesare preferably administered s.c. or i.d. It is also preferred if thevaccine is administered into the skin.

It is believed that uptake of the nucleic acid and expression of theencoded polypeptide by professional antigen presenting cells such asdendritic cells may be the mechanism of priming of the immune response;however, dendritic cells may not be transfected but are still importantsince they may pick up expressed peptide from transfected cells in thetissue (“cross-priming”, e.g., Thomas A M, Santarsiero L M, Lutz E R,Armstrong T D, Chen Y C, Huang L Q, Laheru D A, Goggins M, Hruban R H,Jaffee E M. Mesothelin-specific CD8(+) T cell responses provide evidenceof in vivo cross-priming by antigen-presenting cells in vaccinatedpancreatic cancer patients. J Exp Med. 2004 Aug. 2; 200(3):297-306).

Polynucleotide-mediated immunization therapy of cancer is described inConry et al (1996) Seminars in Oncology 23, 135-147; Condon et al (1996)Nature Medicine 2, 1122-1127; Gong et al (1997) Nature Medicine 3,558-561; Zhai et al (1996) J. Immunol. 156, 700-710; Graham et al (1996)Int J. Cancer 65, 664-670; and Burchell et al (1996) 309-313 In: BreastCancer, Advances in biology and therapeutics, Calvo et al (Eds), JohnLibbey Eurotext, all of which are incorporated herein by reference intheir entireties.

It may also be useful to target the vaccine to specific cellpopulations, for example antigen presenting cells, either by the site ofinjection, use of targeting vectors and delivery systems, or selectivepurification of such a cell population from the patient and ex vivoadministration of the peptide or nucleic acid (for example dendriticcells may be sorted as described in Zhou et al (1995) Blood 86,3295-3301; Roth et al (1996) Scand. J. Immunology 43, 646-651). Forexample, targeting vectors may comprise a tissue- or tumor-specificpromoter which directs expression of the antigen at a suitable place.

Finally, the vaccine according to the invention can be dependent on thespecific type of cancer that the patient to be treated is suffering fromas well as the status of the disease, earlier treatment regimens, theimmune status of the patient, and, of course, the HLA-haplotype of thepatient. Furthermore, the vaccine according to the invention can containindividualised components, according to personal needs of the particularpatient. Examples are different amounts of peptides according to theexpression of the related TAAs in the particular patient, unwantedside-effects due to personal allergies or other treatments, andadjustments for secondary treatments following a first round or schemeof treatment.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from glioblastoma and since it was determined that thesepeptides are not present in normal tissues, these peptides can be usedto diagnose the presence of a cancer.

The presence of the peptides of the present invention on tissue biopsiescan assist a pathologist in diagnosis of cancer. Detection of certainpeptides of the present invention by means of antibodies, massspectrometry or other methods known in the art can tell the pathologistthat the tissue is malignant or inflamed or generally diseased. Presenceof groups of peptides of the present invention can enable classificationor subclassification of diseased tissues.

The detection of the peptides of the present invention on diseasedtissue specimen can enable the decision about the benefit of therapiesinvolving the immune system, especially if T lymphocytes are known orexpected to be involved in the mechanism of action. Loss of MHCexpression is a well described mechanism by which infected or malignantcells escape immunosurveillance. Thus, presence of the peptides of thepresent invention shows that this mechanism is not exploited by theanalyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides of the present invention,such as T cell responses or antibody responses against the peptides ofthe present invention or the peptides of the present invention complexedto MHC molecules. These lymphocyte responses can be used as prognosticmarkers for decision on further therapy steps. These responses can alsobe used as surrogate markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses against thepeptides of the present invention can be considered in the assessment ofside effects. Monitoring of lymphocyte responses might also be avaluable tool for follow-up examinations of transplantation therapies,e.g. for the detection of graft versus host and host versus graftdiseases.

In yet another aspect thereof, the present invention relates to a kitcomprising (a) a container that contains a pharmaceutical composition asdescribed above, in solution or in lyophilized form; (b) optionally, asecond container containing a diluent or reconstituting solution for thelyophilized formulation; and (c) optionally, instructions for (i) use ofthe solution or (ii) reconstitution and/or use of the lyophilizedformulation. The kit may further comprise one or more of (iii) a buffer,(iv) a diluent, (v) a filter, (vi) a needle, or (v) a syringe. Thecontainer is preferably a bottle, a vial, a syringe or test tube; and itmay be a multi-use container. The pharmaceutical composition ispreferably lyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicate directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeated administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, a anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The pharmaceutical formulation of the present invention is one that issuitable for administration of the peptides by any acceptable route suchas oral (enteral), nasal, ophthal, subcutaneous, intradermal,intramuscular, intravenous or transdermal. Preferably the administrationis s.c., and most preferably, i.d. Administration may be by infusionpump.

It should be understood that the features of the invention as disclosedand described herein can be used not only in the respective combinationas indicated but also in a singular fashion without departing from theintended scope of the present invention. For the purposes of the presentinvention, all references as cited herein are incorporated by referencein their entireties.

The invention will now be described in more detail by reference to thefollowing Figures, the Sequence listing, and the Examples. The followingexamples are provided for illustrative purposes only and are notintended to limit the invention.

EXAMPLES 1. Synthesis

Peptides were synthesized by standard and well-established solid phasepeptide synthesis using the Fmoc-strategy. After purification bypreparative RP-HPLC, ion-exchange procedure was performed to incorporatephysiological compatible counter ions (for example acetate, ammonium orchloride). Finally, white to off-white solids were obtained afterlyophilisation. All TUMAPs are preferably administered as acetate salts,other salt-forms are also possible.

Importantly, identity and purity of each individual peptide have beendetermined by mass spectrometry and analytical RP-HPLC. Afterion-exchange procedure the peptides were obtained as white to off-whitelyophilizates in purities shown in table 7:

TABLE 7 PEPTIDE LENGTH PURITY (NO OF AMINO SALT [REL. SEQ ID NO. PEPTIDEID ACIDS) FORM AREA %] 1 ASPM-002 9 ACETATE 92.5 2 BIR-002 15 ACETATE96.3 3 CDC2-001 10 ACETATE 94.8 4 MET-006 9 ACETATE 96.0 5 MMP11-001 10ACETATE 96.1 6 MST1R-001 9 ACETATE 96.3 7 PPAP2C-001 9 ACETATE 94.4 8PROM1-001 9 ACETATE 97.1 9 SMC4-001 9 ACETATE 90.7 10 UCHL5-001 9ACETATE 95.1 11 UQCRB-001 10 ACETATE 97.3 12 (HBV-001) 10 ACETATE 99.520 AVL9-001 9 ACETATE 98.6 24 ERBB3-001 9 ACETATE 99.1

All peptide have been tested with respect to their stability atdifferent physicochemical conditions such as different temperatures andph-values.

2. Components of the Exemplary Pharmaceutical Composition IMA941

IMA941 is composed of a cocktail of synthetic tumor associated peptides(TUMAPs) of which the majority has been identified on primary colorectalcancer cells. The TUMAPs include 10 HLA class I-binding peptides withthe capacity to activate cytotoxic T cells (CD8+ T cells), 1 HLA classII-binding peptide with the capacity to activate T helper cells (CD4+ Tcells) T helper cells play a crucial role in assisting the function ofcytotoxic T cells by releasing cytokines which enhance the killerfunction of CD8+ T cells and may also act directly against tumor cells(Knutson and Disis, 2005). In addition to these 11 TUMAPs IMA941 maycontain one viral control peptide.

Samples from surgically removed malignant and normal tissue from GBMpatients and blood from healthy donors were analyzed in a stepwiseapproach:

First, genome-wide mRNA expression analysis by microarrays was used toidentify genes overexpressed in the malignant tissue compared with arange of normal organs and tissues. In a second step, HLA ligands fromthe malignant material were identified by mass spectrometry.Subsequently identified HLA ligands were compared to gene expressiondata. Peptides encoded by selectively expressed or overexpressed genesas detected in step 1 were considered suitable candidate TUMAPs for amulti-peptide vaccine.

Finally, peripheral CD8+ T cells of healthy individuals were tested forreactivity against the tumor-associated HLA ligands using severalimmunoassays (in vitro T-cell assays).

3. Presentation of Tumor Associated Peptides (TUMAPs) Contained inIMA941 on Tumor Samples Tissue Samples

Patients' tumor tissues were provided by Kyoto Prefectural University ofMedicine (KPUM), Kyoto, Japan, and Osaka City University Graduate Schoolof Medicine (OCU), Osaka, Japan. Written informed consents of allpatients had been given before surgery. Tissues were shock-frozen inliquid nitrogen immediately after surgery and stored until isolation ofTUMAPs at −80° C.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991) (Seeger et al., 1999) using the HLA-A, -B,-C-specific antibody W6/32, CNBr-activated sepharose, acid treatment,and ultrafiltration.

Detection of TUMAPs by ESI-Liquid Chromatography Mass Spectrometry(ESI-LCMS)

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (Acquity HPLC system,Waters) and the eluting peptides were analyzed in an LTQ-Orbitrap hybridmass spectrometer (ThermoElectron) equipped with an ESI source. Peptidepools were loaded directly onto the analytical fused-silicamicro-capillary column (75 μm i.d.×250 mm) packed with 1.7 μm C18reversed-phase material (Waters) applying a flow rate of 400 nL perminute. Subsequently, the peptides were separated using a two-step 180minute-binary gradient from 10% to 33% B at a flow rate of 300 nL perminute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESI source. The LTQ-Orbitrap mass spectrometer was operated inthe data-dependent mode using a TOPS strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30 000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST® and additional manual control. The identified peptide sequencewas assured by comparison of the generated natural peptide fragmentationpattern with the fragmentation pattern of a synthetic sequence-identicalreference peptide. FIG. 1 shows an exemplary spectrum obtained fromtumor tissue for the MHC class I associated peptide CDC2-001 and itselution profile on the UPLC system.

Example 2 Expression Profiling of Genes Encoding the Peptides of theInvention

Not all peptides identified as being presented on the surface of tumorcells by MHC molecules are suitable for immunotherapy, because themajority of these peptides are derived from normal cellular proteinsexpressed by many cell types. Only few of these peptides aretumor-associated and likely able to induce T cells with a highspecificity of recognition for the tumor from which they were derived.In order to identify such peptides and minimize the risk forautoimmunity induced by vaccination the inventors focused on thosepeptides that are derived from proteins that are over-expressed on tumorcells compared to the majority of normal tissues.

The ideal peptide will be derived from a protein that is unique to thetumor and not present in any other tissue. To identify peptides that arederived from genes with an expression profile similar to the ideal onethe identified peptides were assigned to the proteins and genes,respectively, from which they were derived and expression profiles ofthese genes were generated.

RNA Sources and Preparation

Surgically removed tissue specimens were provided by two differentclinical sites (see Example 1) after written informed consent had beenobtained from each patient. Tumor tissue specimens were snap-frozen inliquid nitrogen immediately after surgery and later homogenized withmortar and pestle under liquid nitrogen. Total RNA was prepared fromthese samples using TRI Reagent (Ambion, Darmstadt, Germany) followed bya cleanup with RNEASY® (QIAGEN, Hilden, Germany); both methods wereperformed according to the manufacturer's protocol.

Total RNA from healthy human tissues was obtained commercially (Ambion,Huntingdon, UK; Clontech, Heidelberg, Germany; Stratagene, Amsterdam,Netherlands; BioChain, Hayward, Calif., USA). The RNA from severalindividuals (between 2 and 123 individuals) was mixed such that RNA fromeach individual was equally weighted. Leukocytes were isolated fromblood samples of 4 healthy volunteers.

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

Microarray Experiments

Gene expression analysis of all tumor and normal tissue RNA samples wasperformed by Affymetrix Human Genome (HG) U133A or HG-U133 Plus 2.0oligonucleotide microarrays (Affymetrix, Santa Clara, Calif., USA). Allsteps were carried out according to the Affymetrix manual. Briefly,double-stranded cDNA was synthesized from 5-8 μg of total RNA, usingSuperScript RTII (Invitrogen) and the oligo-dT-T7 primer (MWG Biotech,Ebersberg, Germany) as described in the manual. In vitro transcriptionwas performed with the BioArray High Yield RNA Transcript Labelling Kit(ENZO Diagnostics, Inc., Farmingdale, N.Y., USA) for the U133A arrays orwith the GeneChip IVT Labelling Kit (Affymetrix) for the U133 Plus 2.0arrays, followed by cRNA fragmentation, hybridization, and staining withstreptavidin-phycoerythrin and biotinylated anti-streptavidin antibody(Molecular Probes, Leiden, Netherlands). Images were scanned with theAgilent 2500A GeneArray Scanner (U133A) or the Affymetrix Gene-ChipScanner 3000 (U133 Plus 2.0), and data were analyzed with the GCOSsoftware (Affymetrix), using default settings for all parameters. Fornormalisation, 100 housekeeping genes provided by Affymetrix were used.Relative expression values were calculated from the signal log ratiosgiven by the software and the normal kidney sample was arbitrarily setto 1.0.

The expression profiles of source genes of the present invention thatare highly over-expressed in gastric cancer are shown in FIG. 2.

4. In Vitro Immunogenicity for IMA941 MHC Class I Presented Peptides

To obtain information regarding the immunogenicity of the TUMAPs of thepresent invention, investigations were performed using a wellestablished in vitro stimulation platform already described by (Walter,S, Herrgen, L, Schoor, O, Jung, G, Wernet, D, Buhring, H J, Rammensee, HG, and Stevanovic, S; 2003, Cutting edge: predetermined avidity of humanCD8 T cells expanded on calibrated MHC/anti-CD28-coated microspheres, J.Immunol., 171, 4974-4978). Using this platform, immunogenicity could beshown for the 10 HLA-A*2402 restricted TUMAPs of the invention, thusdemonstrating that these peptides are T-cell epitopes against which CD8+precursor T cells exist in humans (Table 6).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells (aAPC) loaded with peptide-MHC complex (pMHC) andanti-CD28 antibody, we first isolated CD8 T cells from fresh HLA-A*24leukapheresis products of healthy donors obtained from the Blood BankTuebingen.

CD8 T cells were either directly enriched from the leukapheresis productor PBMCs (peripheral blood mononuclear cells) were isolated first byusing standard gradient separation medium (PAA, Cölbe, Germany).Isolated CD8 lymphocytes or PBMCs were incubated until use in T-cellmedium (TCM) consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nürnberg, Germany) were also added to the TCM at this step. Isolation ofCD8+ lymphocytes was performed by positive selection using CD8MicroBeads (Miltenyi Biotec, Bergisch-Gladbach, Germany).

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed as described before (Walter et al., 2003) withminor modifications. Briefly, biotinylated peptide-loaded recombinantHLA-A*2402 molecules lacking the transmembrane domain and biotinylatedat the carboxy terminus of the heavy chain were produced. The purifiedcostimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung et al., 1987) waschemically biotinylated using Sulfo-N-hydroxysuccinimidobiotin asrecommended by the manufacturer (Perbio, Bonn, Germany). Beads used were5.6 μm large streptavidin coated polystyrene particles (BangsLaboratories, Illinois, USA). pMHC used as controls were A*0201/MLA-001(peptide ELAGIGILTV (SEQ ID NO: 25) from modified Melan-A/MART-1) andA*0201/DDX5-001 (YLLPAIVHI (SEQ ID NO: 26) from DDX5), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of600 ng biotin anti-CD28 plus 200 ng relevant biotin-pMHC (high densitybeads). Stimulations were initiated in 96-well plates by co-incubating1×10⁶ CD8+ T cells with 2×10⁵ washed coated beads in 200 μl TCMsupplemented with 5 ng/ml IL-12 (PromoCell) for 3-4 days at 37° C. Halfof the medium was then exchanged by fresh TCM supplemented with 80 U/mlIL-2 and incubating was continued for 3-4 days at 37° C. Thisstimulation cycle was performed for a total of three times. Finally,multimeric analyses were performed by staining the cells withLive/dead-Aqua dye (Invitrogen, Karlsruhe, Germany), CD8-FITC antibodyclone SK1 (BD, Heidelberg, Germany) and PE- or APC-coupled A*2402 MHCmultimers. For analysis, a BD LSRII SORP cytometer equipped withappropriate lasers and filters was used. Peptide specific cells werecalculated as percentage of total CD8+ cells. Evaluation of multimericanalysis was done using the FlowJo software (Tree Star, Oregon, USA). Invitro priming of specific multimer+ CD8+ lymphocytes was detected byappropriate gating and by comparing to negative control stimulations.Immunogenicity for a given antigen was detected if at least oneevaluable in vitro stimulated well of one healthy donor was found tocontain a specific CD8+ T-cell line after in vitro stimulation (i.e.this well contained at least 1% of specific multimer+ among CD8+ T-cellsand the percentage of specific multimer+ cells was at least 10× themedian of the negative control stimulations).

In Vitro Immunogenicity for IMA941 Peptides

For tested HLA class I peptides, in vitro immunogenicity could bedemonstrated for all 14 peptides by generation of peptide specificT-cell lines. Exemplary flow cytometry results after TUMAP-specificmultimer staining for two peptides of the invention are shown in FIG. 3,together with a corresponding negative control. Results for the peptidesfrom the invention are summarized in Table 8, which provides in vitroimmunogenicity of HLA class I peptides of the invention. The results ofin vitro immunogenicity experiments show the percentage of positivetested donors and wells among evaluable peptides. At least two donorsand 24 wells were evaluable for each peptide.

TABLE 8 In vitro immunogenicity of HLA class I peptides of the inventionPositive donors/ Positive wells/ Antigen donors tested [%] wells tested[%] SEQ ID No CDC2-001 88 28 1 ASPM-002 63 31 2 MET-006 63 22 4UCHL5-001 75 14 3 MST1R-001 50 14 7 SMC4-001 75 9 9 PROM1-001 83 26 5MMP11-001 33 11 10 ABL1-001 50 13 21 AVL9-001 100 50 20 NUF2-002 50 4 22NUF2-001 100 25 19 PPAP2C-001 100 54 8 UQCRB-001 100 38 6 ERBB3-001 8315 24

5. Immunogenicity of IMA941 Class II TUMAP BIR-002

A clinical study was conducted in order to confirm the immunogenicity ofthe peptide with the SEQ ID NO:11.

The primary study objective was the investigation of the PSA(prostate-specific antigen)-based response (PSA-R) to the subcutaneousadministration of a prostate-specific peptide panel (vaccinationtherapy) in patients with biochemical relapse after radicalprostatectomy without detection of manifest metastatic lesions.

The secondary study objective was the investigation of the tolerabilityand feasibility of administering vaccination therapy in patients withprostate carcinoma with special consideration of immunological phenomenain terms of a T cell response.

The study was designed as a prospective, randomized Phase I/II study forthe indication of “biochemical relapse after radical prostatectomywithout detection of manifest metastatic lesions.”

Study Population

As part of this Phase I/II study, an attempt was made to induce PSAregression as an indicator of cessation of tumor growth by means ofvaccination with a prostate-specific peptide panel in HLA-A*02⁺ patientswith biochemical relapse after radical prostatectomy. A combination ofprostate-specific peptides was administered subcutaneously withevaluation of the extent of the respective immune response in thecontext of various administration forms of the antigenic structures.

In contrast to previous vaccination studies, the planned study targetedthe treatment of patients with a small tumor burden not yet detectableby imaging procedures. The patients were all immunized in the same wayusing known prostate-specific antigenic structures to enhance the immuneresponse to the malignantly transformed cells. Nineteen patients weretreated.

TABLE 9 Characteristics of study population Total % Median Range Age 1963 55-77 Prior neo-/adjuvant treatment None 11 58 Radiation 3 16Intermittent Hormonal 2 11 Therapy Rad. + Int. Horm. Therapy 2 11 Rad. +Chemotherapy 1 5 TNM at RPX T2a-c R0 6 32 T3a-c R0 6 32 T2a-c R1 3 16T3a-c R1 3 16 T3aN2 R0 1 5 Gleason score 5-7 10 53 8-10 3 16 unknown 632 RPX prior to vaccination in 41  9-124 months First relapse post OP inmonths 14  1-90 PSA at vaccination start 0.76 0.14-10.8Treatment Plan

After rule-out of manifest metastatic lesions using computed tomographyand skeletal scintigraphy, the prostate-specific peptide vaccine wassubcutaneously administered according to the different administrationforms to patients with detected PSA relapse after prior radicalprostatectomy (PSA increase in terms of a 50% elevated value during twomeasurements at least 14 days apart). The vaccine was administered 8× ondays 0, 7, 14, 28, 42, and 56 (approximately 100 micrograms per peptideand injection each time). After each vaccination treatment and again onday 70, PSA was measured to evaluate the therapeutic response.

If a tumor response (complete remission [PSA-CR], partial remission[PSA-PR], or stable clinical course [no change, PSA-NC]) is detected,the patient received the vaccine once a month as maintenance therapyaccording to the selected administration form. The patient's response tovaccination therapy was evaluated in detail as follows:

Complete remission (PSA-CR): Normalization of an initially elevated PSAlevel, confirmed by measurement after an interval of at least 4 weeks.Normalization is defined as a PSA nadir of <0.2 ng/ml, which would beexpected after radical prostatectomy with complete tumor or prostateextirpation.

Partial remission: a) PSA-PR≦80% (Reduction in an initially elevated PSAlevel by 80%, confirmed by measurement after an interval of at least 4weeks); and b) PSA-PR≦50% (Reduction in an initially elevated PSA levelby 50%, confirmed by measurement after an interval of at least 4 weeks.)

Stable disease (PSA-SD): No significant change over a period of at leastfour weeks. This includes stabilization and a reduction of less than 50%and an increase of less than 10%, confirmed by measurement after aninterval of at least 4 weeks.

Progression (PSA-PD): Increase in the PSA level by more than 10%. In theevent of PSA progression, the study was terminated.

After enrollment of the patients into the study, the epitope-specificvaccine was used; the proteins specifically expressed in prostaticepithelial cells (e.g., PSMA/PSCA) were taken into account. In additionto investigating the general efficacy of the administered vaccine withrespect to monitoring the growth of residual tumor fractions asevaluated by PSA monitoring, this study investigated the effects ofvarious vaccination methods with respect to efficient modulation of theimmune system. In addition to simple subcutaneous administration of thepeptides alone, various combinations with adjuvants were also used. Inparticular, depot and adjuvant activity for peptide vaccines ofMontanide (a formulation of the classical incomplete Freund's adjuvantsuitable for use in humans), which has recently been described veryfavorably, was used. For this purpose, 500 μl of the peptide solutionwas mixed with 500 μl of Montanide and administered. Thereby, awater-in-oil emulsion is built that slowly releases the antigencontained in the aqueous phase over weeks. The physical stability of theemulsion is very high, as at 4° C. it can be stored for more than 3months without significant phase separation. The depot function ofMontanide has been exploited in several vaccination trials with goodresults (Oka et al., 2004).

One study arm investigated the efficacy of vaccination duringconcomitant stimulation of the immune system by growth factors, GM-CSF,Leukine® solution for injection. GM-CSF is a very commonly used adjuvantin peptide vaccination trials with several thereof reporting enhancedclinical and T-cell responses. Initially, GM-CSF is a dendritic cellrecruitment and differentiation factor that is thought to enhance thenumber of dendritic cells at the vaccines' injection site. AlthoughGM-CSF does not by itself activate antigen presenting cells as dendriticcells and macrophages an indirect activation in vivo has been reported(Molenkamp et al., 2005).

Another study arm investigated the efficacy of vaccination duringconcomitant activation of dendritic cells by epicutaneous use ofimiquimod. Imiquimod was administered as an 5% ointment (Aldara). It hasa strong immunostimulatory via its effect on TLR7 positive cells (e.g.plasmacytoid DCs, Langerhans cells, dermal DCs) and activates theMyD88-dependent pathway. Activated APCs release T-cell stimulating andinflammatory cytokines, upregulate costimulation and migrate to draininglymph nodes. The potential of imiquimod to enhance peptide-induced CTLresponse by mixing the antigens into the ointment or by application ofAldara over the s.c. or i.d. injection site for the antigens has beendemonstrated in animal models.

Another study arm investigated the efficacy of vaccination duringconcomitant activation of dendritic cells by mixing them withprotamine-stabilized mRNA encoding mucin-1 to activate TLR 7/8. mRNAshows a broad activation of mouse and human immune cell populations. Thepresence of the poly-basic protein protamine in the formulationincreases the half-life of the mRNA and induces the formation ofpotentially depot-forming particles. This adjuvant may therefore combinedepot-forming and APC-activating properties.

In summary, the administration forms of the vaccine included thefollowing approaches:

-   -   Subcutaneous administration of the peptide vaccine emulsified in        Montanide,    -   Subcutaneous administration of the peptide vaccine emulsified in        500 μl of Montanide in combination with topical administration        of 225 μl of GM-CSF with the objective of achieving a stronger        immune response triggered by concomitant administration of        growth factors,    -   Subcutaneous administration of the peptide vaccine emulsified in        500 μl of Montanide in combination with local hyperthermia, the        latter given with the objective of achieving a thermally induced        stronger immune response,    -   Subcutaneous administration of the peptide vaccine emulsified in        500 μl of Montanide in combination with epicutaneous imiquimod        in order to activate dendritic cells via TLR 7,    -   Subcutaneous administration of the peptide vaccine emulsified in        500 μl of Montanide together with 55 μl of mucin-1        mRNA/protamine in order to activate dendritic cells via TLR 7/8.        Schedule: The entire duration of the study was 3 years.

Prostate-specific peptide vaccines were administered to patients on days0, 7, 14, 28, 42, and 56. In patients with stable disease or anobjective tumor response (PSA-CR or PSA-PR), the vaccinations werereceived once a month i.d. until detectable progression occurred. On thebasis of the experience available thus far, peptide injections aretolerated without significant adverse reactions. Because the response tovaccination therapy was evaluated solely serologically on the basis ofthe PSA measurement, a test was performed at the start of the study todetermine whether the administered vaccine interferes with PSAmeasurement in vitro, which could simulate a clinical response. On days0, 7, 14, 28, 42, 56, and 70, blood samples were taken for laboratorytests, PSA levels, differential blood count, FACS analysis, andcytokines. If treatment is continued past day 70, 6-week PSA monitoringwas performed in order to detect treatment failure in a timely manner.

Treatment was ended if documented progression of the disease occurred interms of a continuous PSA elevation.

Beginning on day 84, immunization therapy was continued at 4-weekintervals until documented progression or up to day 420 (15 months).Decisions regarding continuation of therapy (in successful cases)outside of this study were made on a case-by-case basis. Unexpectedadverse reactions did not occur in this study.

The laboratory tests included coagulation, electrolytes, LDH, β2-M, CK,hepatic enzymes, bilirubin, creatinine, uric acid, total protein,coagulation, CRP, differential blood count with smear, PSA level,cytokines, FACS, Elispot.

Analysis of the cutaneous reaction to defined bacterial and fungalantigens (48-72 hours after administration, delayed typehypersensitivity (DTH), T cell-mediated, will serve as an analysis ofthe patient's cellular immune system before the start of the study).

The peptides required for the study (nona-peptides) were manufactured inthe laboratory of PD Dr. Stefan Stevanovic in the department of Prof.H.-G. Rammensee. These peptides were purified by HPLC and analyzed bymass spectrometry. The purity of the peptides can also be checked byHPLC, mass spectrometry, and Edman sequencing. Using these methods,purity of up to 98% can be documented (which must be regarded as themaximum according to the current state of the methods). The synthesizedpeptides were dissolved in DMSO (CryoSure, WAK Chemie Medical GmbH; 10mg/ml), diluted to 1:10 in Ampuwa (Fresenius Kabi), and aliquoted understerile conditions.

Clinical Response

In two patients PET-CT scan could reveal local recurrence after localtumor was detected by continuous digital rectal examination. In theremaining 17 patients the location of disease activity could not beverified at study termination.

Repeated laboratory evaluation of differential blood count or extensiveclinical chemistry did not reveal any abnormalities or changes duringthe study.

Of the 19 patients 16 patients reacted to the Survivin II peptide (IFN-gELISPOT, +/−ICS) according to SEQ ID NO:12. Among them, were 12 patientswith induction of an anti-survivin T-cell response upon vaccination, 2with pre existing anti-Survivin T cells and 2 patients of whom it wasnot determined, whether pre existing anti-Survivin T cells wereabundant.

Biochemical Response

Complete response was considered as a non-detectable PSA value accordingto the lowest value detectable of the laboratory collaborating afterinitially elevated PSA. The measurement had to be confirmed after anintervall of at least four weeks. A PR>80% and >50% had to bereevaluated after four weeks accordingly. A PSA within the range of lessthan 50% decrease or less than 10% increase reflected stable disease ifat least confirmed after four weeks. Progressive disease was consideredany increase of more than 10% of PSA at treatment start.

Biochemical response in patients who terminated the study was followeduntil they received further treatment with local radiation orantihormonal therapy.

19 patients consented to participate and the data was analyzed with thelongest follow-up lasting about 3.75 years.

PSA Stability and DT Increase

PSA values of two patients (10.2%) exhibited stability according to theabove mentioned criteria of biochemical response which state that norise of the PSA value greater than 10% at treatment start had occurredat study end (FIG. 6, Tables 10, 11, and 12). Follow up in those twocases was conducted 14 and 16 months after the last vaccine application.Average duration of stability was 24 months (28 and 31) at data cut-offwith an average of 18 vaccinations (14 and 20) applied.

One of these two patients showed partial response >50% for a period of 9months, followed by a period of slow PSA rise with a doubling time of20.5 compared to 9.8 months prior vaccination. Initial PSA relapsestarted 18 months post surgery for a pT2pN0 Gleason 5 tumor.

At data analysis Patient 8 exhibited stable disease since the beginningof the vaccination program 28 months ago. He had stopped treatment dueto an allergic reaction after 10 months and the 14^(th) vaccination. Hehad an unfavorable pT3b Gleason 3+4 situation with a PSA nadir afterradical prostatectomy not below 0.6 ng/ml and PSA progression withouttimely delay after initial decline postoperatively. Doubling time slowedfrom 6.6 months to 148 months.

These two patients received dermal Imiquimod at the application site ateach peptide vaccination.

PSA DT Increase without PSA Stability

PSA DT of patient 11 was increased from 1.5 to 10.1 months during sixmonth on study. Since he started with a PSA of 10.8 ng/ml and progressedto 17.8 ng/ml he terminated study procedures to receive antiandrogenmonotherapy without any malignant lesions visualized in PET-CT. Hereceived Aldara as adjuvant.

Patient 16 started into vaccine treatment plus Mucin-1-mRNA/protaminewith a doubling time of 6.1 months. PSA velocity declined into a halflife time of 2.7 months for five months followed by a statisticallycalculated rise of PSA DT of 14.4 months which is continuing 16 monthsafter treatment start. With an initial PSA of 0.29 ng/ml, he dropped to0.19 ng/ml during the first 5 months on study treatment, rose to 0.4ng/ml within the following 8 months and terminated the study perprotocol with 0.41 ng/ml 19 months after treatment start.

PSA Progression

Patient 5 progressed during the study according to the estimated PSAdoubling time before vaccination. However, he experienced a PSA declinewith a half-time life of 20.2 months after treatment end for acontinuing period of 10 months at data cut-off. He still was notreceiving any secondary treatment after vaccination end. He wasvaccinated with montanide as the only adjuvant.

TABLE 10 PSA Doubling Time in months Geometric Range of Total % Mean DTPSA DT prior vaccination in months 19  8.3 1.5-44.8 PSA DT at study endor at end 18* 11.2 2.2-148  of follow-up No change of PSA DT during 11 58 2.2-44.8 vaccination Increased PSA DT continuing at 4 21 end of studyNo change of PSA DT during vacc 1 5 but decline after Interim PSAdecline or DT increase 3 16 followed by DT decrease *PSA DT at study endor end of follow-up was not included for Pat. 5 due to PSA decline

7. Binding of HLA class I-Restricted Peptides of the Invention toHLA-A*0201 Objective and Summary

The objective of this analysis was to evaluate the affinity of the HLAclass I peptides to the MHC molecule coded by the HLA-A*0201 allele asthis is an important parameter for the mode of action of IMA941.Affinities to HLA-A*0201 were medium to high for all 10 HLA classI-restricted peptide in IMA941 and MET-001, dissociations constants (KD)being in the range from 0.14 (MET-001) to 2.05 nM (CSP-001). All valuesare in the range between 0.1 for the strong binder HBV-001 and 4.4 forthe intermediate binder MUC-001. These results confirmed the strongbinding affinity of all HLA class I peptides of the IMA941 vaccinecandidate and the MET-005 derived MET-001 to HLA-A*02.

Principle of Test

Stable HLA/peptide complexes consist of three molecules: HLA heavychain, beta-2 microglobulin (b2m) and the peptidic ligand. The activityof denatured recombinant HLA-A*0201 heavy chain molecules alone can bepreserved making them functional equivalents of “empty HLA-A*0201molecules”. When diluted into aqueous buffer containing b2m and anappropriate peptide, these molecules fold rapidly and efficiently in anentirely peptide-dependent manner. The availability of these moleculesis used in an ELISA-based assay to measure the affinity of interactionbetween peptide and HLA class I molecule (Sylvester, 2002).

Purified recombinant HLA-A*0201 molecules were incubated together withb2m and graded doses of the peptide of interest. Instead of full-lengthMET-005 that does not possess HLA class I binding capacities, the provenA*0-binding product MET-001 was included into the analysis that isgenerated in vivo from MET-005 by naturally occurring antigenprocessing. The amount of de novo-folded HLA/peptide complexes wasdetermined by a quantitative ELISA. Dissociation constants (KD values)were calculated using a standard curve recorded from dilutions of acalibrant HLA/peptide complex.

Results

Results are shown in FIG. 2. A lower KD value reflects higher affinityto HLA-A*0201. Most of the IMA941 peptides had similar and strongaffinities to HLA-A*0201 within the range from 0.1 (HBV-001, strongbinder) to 44.4 nM (MUC-001, intermediate binder). Thereby, all IMA941class I TUMAPs have a medium to strong binding affinity to the MHCmolecule A*02.

8. Binding of HLA Class II-Restricted Peptides of the Invention toHLA-DR Objective and Summary

Class II TUMAPs activate helper T cells which play a crucial role inassisting the function of CTLs triggered by class I-restricted TUMAPs.Binding of the IMA941 class II peptides to several different HLA classII molecules (promiscuous binding) is important to ensure that themajority of patients treated with the vaccine candidate IMA941 are ableto benefit from a supportive helper T cell response. HLA-DR for example,the most dominantly expressed human HLA class II molecule, is highlypolymorphic with several hundreds of known alleles. Based on knownallele frequencies for HLA-DRB1 haplotypes and well-established bindingalgorithms, it can be predicted that both HLA class II ligands inIMA941—IMA-BIR-002 and IMA-MET-005—are promiscuous HLA-DR bindingpeptides. In detail, the probability that an HLA-A*02-positive Caucasianexpresses at least one suitable HLA-DR allele is >90% for both IMA941class II TUMAPs. As the remaining human class II alleles HLA-DQ and -DPwere omitted from this calculation due to the lack of frequency data orbinding prediction algorithms, the real promiscuity is most likely evenhigher. The calculated promiscuity of the two IMA941 class II TUMAPs isin the same range as for the known pan-DR epitope (PADRE, genotypicfrequency Fprojected=93.1%). In addition, the promiscuous binding ofthese peptides was confirmed experimentally by in vitro binding assays.Moreover, for IMA-BIR-002 a high in vivo immunogenicity could bedemonstrated (see above). Summarizing, these results confirm thatMET-005 and BIR-002 are promiscuous HLA-DR binding peptides.

Principle of Binding Prediction

Using the SYFPEITHI algorithm developed at the University of Tübingen(Rammensee et al., 1997; Rammensee et al., 1999), binding of IMA941class II TUMAPs to several common HLA-DR alleles was ranked. Thealgorithm has already been successfully used to identify class I andclass II epitopes from a wide range of antigens, e.g. from the humantumor-associated antigens TRP2 (class I) (Sun et al., 2000) and SSX2(class II) (Neumann et al., 2004). The threshold for binding was definedat a score of 18 based on the analysis of binding scores of knownpublished promiscuous HLA-DR ligands.

Published HLA-DR haplotype frequencies among the HLA-A*02 positiveCaucasian population (Mori et al., 1997) and frequencies ofhigh-resolution haplotypes (Chanock et al., 2004) were used (see Table2). The haplotype frequency is the frequency of a distinct allele on anindividual chromosome. Due to the diploid set of chromosomes withinmammalian cells, the frequency of genotypic occurrence of this allele ishigher and can be calculated employing the Hardy-Weinberg principle(haplotype frequency G_(f) results in a genotypic occurrence F(F=2G_(f)−G_(f) ²]).

The sum of frequency of DRB1-haplotypes with known SYFPEITHI matrix andknown individual frequency among the A*02+ Caucasian population is47.8%. Therefore, the predicted binding distribution of class II TUMAPsto these alleles was projected to the remaining 52.2% of DRB1-allelesfor which these data are not available.

Finally, promiscuous binding is defined as binding of a peptide toseveral HLA-DR alleles with the probability that one of these isexpressed in the Caucasian population being at least 50%.

Principle of In Vitro Binding Assay (ProImmune REVEAL™)

IMA-BIR-002 and IMA-MET-005 were assembled with HLA-DR broad antigens(HLA-DR1 to DR7, which comprise also the split antigens HLA-DR11 to-DR15 (Mori et al., 1997)) and analyzed using the REVEAL™ MHC:peptidebinding assay (ProImmune, Oxford, UK) to determine their level ofincorporation into MHC molecules. In this assay, binding was compared tothat of a pass/fail control binder, and to a positive control peptidefor each HLA-DR antigen.

Results

Based on the prediction by the SYFPEITHI algorithm IMA-BIR-002 is likelyto bind to ⅞ of HLA-DR alleles with known binding motif (Table 11). Theprobability that an HLA-A*02 positive Caucasian expresses at least onesuitable HLA-DRB1 allele for IMA-BIR-002 is 92.6%. Therefore, bothIMA941 class II peptides are predicted to be promiscuous HLA-DR binders.

If the haplotype frequency of binding HLA-DRB1 alleles was overestimatedthrough this approach by factor two, their genotypic occurrence wouldstill be >50% for all class II TUMAPs in IMA941. In addition,experimental confirmation for promiscuous binding of IMA-BIR-002 toHLA-DR1, 3, 4 and 11 was obtained from in vitro binding data (FIG. 3).

As IMA-BIR-002 has proven broad immunogenicity in a clinical trial inprostate cancer patients with different HLA-DR alleles, thepromiscuitivity of this class II peptide has clearly been proven invivo.

In conclusion, in silico analysis of the HLA-DR binding properties ofthe two class II peptides contained in IMA941 and additionalexperimental evidence from in vitro assays and from a clinical trialwith BIR-002 strongly suggest that these TUMAPs are promiscuous bindersof human class II HLA molecules.

TABLE 11 Binding scores of IMA941 class II TUMAPs to HLA-DR alleles withknown binding motif. Shown are the SYFPEITHI binding scores for the mostcommon HLA-DRB1 alleles in the Caucasian population. p gives thehaplotype frequencies among HLA-A*02 positive Caucasians. The peptidewas considered as binding to an HLA molecule if the score was equal toor higher than 18. Accumulation of the p values for binding DRB1 allelesresults in the minimal haplotype frequency p_(min). Extrapolation ofthese frequencies to all DRB1 alleles including those with incompletebinding prediction matrix or frequency data gives the projectedhaplotype frequency p_(projected) that corresponds to the frequency ofgenotypic occurrence F_(projected). n.d. = no data IMA-BIR-002 DRB1*allele 0101 0301 0401 0404 0701 1101 1104 1501 SYFPEITHI score 28 29 2824 14 32 24 30 p 6.6% 5.9% 9.6% 6.0% 13.0% 4.4% 2.3% n.d. predictedbinding yes yes yes yes no yes yes yes p_(min) 34.8% Haplotypicfrequency p_(projected) 72.8% Genotypic frequency F_(projected) 92.6%

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The invention claimed is:
 1. A pharmaceutical composition, comprising atleast two peptides, wherein said at least two peptides are HLA-A*024ligands consisting of an amino acid sequence according to SEQ ID NO: 1and SEQ ID NO: 2, and wherein said peptides are in the form of apharmaceutically acceptable salt.
 2. The pharmaceutical composition ofclaim 1, wherein at least one peptide includes non-peptide bonds.
 3. Thepharmaceutical composition according to claim 1 wherein the selection,number or amount of peptides present in the composition is tissue-,cancer-, or patient-specific.
 4. The pharmaceutical compositionaccording to claim 1 further comprising at least one suitable adjuvant.5. The pharmaceutical composition according to claim 4, wherein theadjuvant is a colony-stimulating factor.
 6. The pharmaceuticalcomposition according to claim 5, wherein the colony-stimulating factorsis selected from the group consisting of Granulocyte Macrophage ColonyStimulating Factor (GM-CSF), imiquimod, and resimiquimod.
 7. Thepharmaceutical composition according to claim 1, additionally containingat least one antigen presenting cell.
 8. The pharmaceutical compositionaccording to claim 7, wherein the antigen presenting cell is a dendriticcell.
 9. The pharmaceutical composition according to claim 7, whereinthe at least one antigen presenting cell a) is pulsed or loaded with thepeptide or b) comprises an expression construct encoding the peptide.10. The pharmaceutical composition of claim 1, wherein thepharmaceutical composition is administered intravenously,intra-arterially, intra-peritoneally, intramuscularly, intradermally,intratumorally, orally, dermally, nasally, buccally, rectally,vaginally, by inhalation, or by topical administration.